Compositions and methods for insecticidal control of stinkbugs

ABSTRACT

Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a  Pentatomidae  plant pest or a  N. viridula, Acrosternum hilare, Piezodorus guildini , and/or  Halymorpha halys  plant pest, decrease the expression of a target sequence in the pest. In specific embodiments, the decrease in expression of the target sequence controls the pest and thereby the methods and compositions are capable of limiting damage to a plant. The present invention provides various target polynucleotides set forth in any one of SEQ ID NOS: 1-292 or 302-304 or active variants and fragments thereof, wherein a decrease in expression of one or more the sequences in the target pest controls the pest (i.e., has insecticidal activity). Further provided are silencing elements which when ingested by the pest decrease the level of the target polypeptide and thereby control the pest. In specific embodiment, the pest is  Pentatomidae . Plants, plant part, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof of the invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Non-Provisional applicationSer. No. 14/525,482 filed Oct. 28, 2014, which is a continuation of U.S.Non-Provisional application Ser. No. 13/152,795 filed Jun. 3, 2011, nowgranted as U.S. Pat. No. 8,872,001, which claims the benefit U.S.Provisional Application No. 61/351,405, filed Jun. 4, 2010; the contentsof which are herein incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 3548USDIV_SeqList.txt, a creation date of Jun. 2, 2011 and asize of 199 KB. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

FIELD OF THE INVENTION

The present invention relates generally to methods of molecular biologyand gene silencing to control pests.

BACKGROUND OF THE INVENTION

Insect pests are a serious problem in agriculture. They destroy millionsof acres of staple crops such as corn, soybeans, peas, and cotton.Yearly, these pests cause over $100 billion dollars in crop damage inthe U.S. alone. In an ongoing seasonal battle, farmers must applybillions of gallons of synthetic pesticides to combat these pests. Othermethods employed in the past delivered insecticidal activity bymicroorganisms or genes derived from microorganisms expressed intransgenic plants. For example, certain species of microorganisms of thegenus Bacillus are known to possess pesticidal activity against a broadrange of insect pests including Lepidoptera, Diptera, Coleoptera,Hemiptera, and others. In fact, microbial pesticides, particularly thoseobtained from Bacillus strains, have played an important role inagriculture as alternatives to chemical pest control. Agriculturalscientists have developed crop plants with enhanced insect resistance bygenetically engineering crop plants to produce insecticidal proteinsfrom Bacillus. For example, corn and cotton plants geneticallyengineered to produce Cry toxins (see, e.g., Aronson (2002) Cell Mol.Life Sci. 59(3):417-425; Schnepf et al. (1998) Microbiol. Mol. Biol.Rev. 62(3):775-806) are now widely used in American agriculture and haveprovided the farmer with an alternative to traditional insect-controlmethods. However, these Bt insecticidal proteins only protect plantsfrom a relatively narrow range of pests. Moreover, these modes ofinsecticidal activity provided varying levels of specificity and, insome cases, caused significant environmental consequences.

Previous control of stinkbugs relied on broad spectrum insecticides.With the adoption of transgenic controls for major lepidopteran pests inseveral crops, these insecticides are no longer used and stinkbugs havebecome a major secondary pest. No successful use of transgenic controlof stinkbugs has been described or adopted. This may be due in part tothe extra oral digestion employed by stinkbugs where digestive enzymesare injected into the host plant prior to feeding. This makes itdifficult to find proteins that survive long enough to manifest activityagainst these insects. RNAi may overcome that feeding behavior byrelying on double stranded RNAs rather than proteins. Thus, there is animmediate need for alternative methods to control pests.

BRIEF SUMMARY OF THE INVENTION

Methods and compositions are provided which employ a silencing elementthat, when ingested by a pest, such as a Pentatomidae plant pestincluding for example, a N. viridula (southern green stink bug),Acrosternum hilare (green stinkbug), Piezodorus guildini (redbandedstinkbug), and/or Halymorpha halys (Brown marmorated stinkbug). plantpest, is capable of decreasing the expression of a target sequence inthe pest. In specific embodiments, the decrease in expression of thetarget sequence controls the pest and thereby the methods andcompositions are capable of limiting damage to a plant. The presentinvention provides various target polynucleotides as set forth in SEQ IDNOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 302, 303 or 304 or active variants or fragments thereof,wherein a decrease in expression of one or more the sequences in thetarget pest controls the pest (i.e., has insecticidal activity). Furtherprovided are silencing elements, which when ingested by the pest,decrease the level of expression of one or more of the targetpolynucleotides. Plants, plant parts, plant cells, bacteria and otherhost cells comprising the silencing elements or an active variant orfragment thereof are also provided.

In another embodiment, a method for controlling a pest, such as aPentatomidae plant pest, such as, for example, a N. viridula,Acrosternum hilare, Piezodorus guildini, and/or Halymorpha halys plantpest, is provided. The method comprises feeding to a pest a compositioncomprising a silencing element, wherein the silencing element, wheningested by the pest, reduces the level of a target sequence in the pestand thereby controls the pest. Further provided are methods to protect aplant from a pest. Such methods comprise introducing into the plant orplant part a silencing element of the invention. When the plantexpressing the silencing element is ingested by the pest, the level ofthe target sequence is decreased and the pest is controlled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Southern Green Stinkbug feeding assay results withsoybean embryo tissue transformed with hairpin RNA silencing contructs.

FIG. 2 shows the Southern Green Stinkbug feeding assay results withsoybean embryo tissue transformed with amiRNA silencing constructs.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Overview

Frequently, RNAi discovery methods rely on evaluation of known classesof sensitive genes (transcription factors, housekeeping genes etc.). Incontrast, the target polynucleotide set forth herein were identifiedbased solely on high throughput screens of a library of over 1000expressed sequence tags from N. viridula. This screen allowed for thediscovery of many novel sequences, many of which have extremely low orno homology to known sequences. This method provided the advantage ofhaving no built in bias to genes that are frequently highly conservedacross taxa. As a result, many novel targets for RNAi as well as knowngenes not previously shown to be sensitive to RNAi have been identified.

As such, methods and compositions are provided which employ a silencingelement that, when ingested by a pest, such as a Pentatomidae plant pestor, for example, a N. viridula, Acrosternum hilare, Piezodorus guildini,and/or Halymorpha halys plant pest, is capable of decreasing theexpression of a target sequence in the pest. In specific embodiments,the decrease in expression of the target sequence controls the pest andthereby the methods and compositions are capable of limiting damage to aplant or plant part. The present invention provides targetpolynucleotides as set forth in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 302, 303, or 304.or active variants and fragments thereof. Silencing elements designed inview of these target polynucleotides are provided which, when ingestedby the pest, decrease the expression of one or more of the targetsequences and thereby controls the pest (i.e., has insecticidalactivity).

As used herein, by “controlling a pest” or “controls a pest” is intendedany affect on a pest that results in limiting the damage that the pestcauses. Controlling a pest includes, but is not limited to, killing thepest, inhibiting development of the pest, altering fertility or growthof the pest in such a manner that the pest provides less damage to theplant, decreasing the number of offspring produced, producing less fitpests, producing pests more susceptible to predator attack, or deterringthe pests from eating the plant.

Reducing the level of expression of the target polynucleotide or thepolypeptide encoded thereby, in the pest results in the suppression,control, and/or killing the invading pathogenic organism. Reducing thelevel of expression of the target sequence of the pest will reduce thedisease symptoms resulting from pathogen challenge by at least about 2%to at least about 6%, at least about 5% to about 50%, at least about 10%to about 60%, at least about 30% to about 70%, at least about 40% toabout 80%, or at least about 50% to about 90% or greater. Hence, themethods of the invention can be utilized to control pests, particularly,Pentatomidae plant pest or a N. viridula, Acrosternum hilare, Piezodorusguildini, and/or Halymorpha halys plant pest.

Assays that measure the control of a pest are commonly known in the art,as are methods to quantitate disease resistance in plants followingpathogen infection. See, for example, U.S. Pat. No. 5,614,395, hereinincorporated by reference. Such techniques include, measuring over time,the average lesion diameter, the pathogen biomass, and the overallpercentage of decayed plant tissues. See, for example, Thomma et al.(1998) Plant Biology 95:15107-15111, herein incorporated by reference.See, also Baum et al. (2007) Nature Biotech 11:1322-1326 and WO2007/035650 which proved both whole plant feeding assays and corn rootfeeding assays. Both of these references are herein incorporated byreference in their entirety. See, also the examples below.

The invention is drawn to compositions and methods for protecting plantsfrom a plant pest, such as Pentatomidae plant pests or N. viridula,Acrosternum hilare, Piezodorus guildini, and/or Halymorpha halys plantpests or inducing resistance in a plant to a plant pest, such asPentatomidae plant pests or N. viridula, Acrosternum hilare, Piezodorusguildini, and/or Halymorpha halys plant pests. As used herein“Pentatomidae plant pest” is used to refer to any member of thePentatomidae family. Accordingly, the compositions and methods are alsouseful in protecting plants against any Pentatomidae plant pestincluding representative genera and species such as, but not limited to,Acrocorisellus (A. serraticollis), Acrosternum (A. adelpha, A. hilare,A. herbidum, A. scutellatum), Agonoscelis (A. nubila), Alcaeorrhynchus(A. grandis, A. phymatophorus), Amaurochrous (A. brevitylus), Apateticus(A. anatarius, A. bracteatus, A. cynicus, A. lineolatus, A.marginiventris), Apoecilus, Arma (A. custos), Arvelius, Bagrada, Banasa(B. calva, B. dimiata, B. grisea, B. induta, B. sordida), Brochymena (B.affinis, B. cariosa, B. haedula, B. hoppingi, B. sulcata), Carbula (C.obtusangula, C. sinica), Chinavia, Chlorochroa (C. belfragii, C. kanei,C. norlandi, C. senilis, C. viridicata), Chlorocoris (C. distinctus, C.flaviviridis, C. hebetatus, C. subrugosus, C. tau), Codophila (C.remota, C. sulcata, C. varius), Coenus (C. delius, C. inermis, C.tarsalis), Cosmopepla (C. bimaculata, C. binotata, C. carnifex, C.decorata, C. intergressus), Dalpada (D. oculata), Dendrocoris (D.arizonesis, D. fruticicola, D. humeralis, D. parapini, D. reticulatus),Dolycoris (D. baccarum (sloe bug)), Dybowskyia (D. reticulata), Edessa,Erthesina (E. fullo), Eurydema (E. dominulus, E. gebleri (shield bug),E. pulchra, E. rugosa), Euschistus (E. biformis, E. integer, E.quadrator, E. serous, E. tristigma), Euthyrhynchus (E. floridanus, E.macronemis), Gonopsis (G. coccinea), Graphosoma (G. lineatum (stinkbug), G. rubrolineatum), Halyomorpha (H. halys (brown marmorated stinkbug)), Halys (H. sindillus, H. sulcatus), Holcostethus (H. abbreviatus,H. fulvipes, H. limbolarius, H. piceus, H. sphacelatus), Homalogonia (H.obtusa), Hymenarcys (H. aequalis, H. crassa, H. nervosa, H. perpuncata,H. reticulata), Lelia (L. decempunctata), Lineostethus, Loxa (L.flavicollis, L. viridis), Mecidea (M. indicia, M. major, M. minor),Megarrhamphus (M. hastatus), Menecles (M. insertus, M. portacrus),Mormidea (M. cubrosa, M. lugens, M. parva, M. pictiventris, M. ypsilon),Moromorpha (M. tetra), Murgantia (M. angularis, M. tessellata, M.varicolor, M. violascens), Neottiglossa (N. californica, N. cavifrons,N. coronaciliata, N. sulcifrons, N. undata), Nezara (N. smaragdulus, N.viridula (southern green stink bug)), Oebalus (O. grisescens, O.insularis, O. mexicanus, O. pugnax, O. typhoeus), Oechalia (O.schellenbergii (spined predatory shield bug)), Okeanos (O.quelpartensis), Oplomus (O. catena, O. dichrous, O. tripustulatus),Palomena (P. prasina (green shield bug)), Parabrochymena, Pentatoma (P.angulata, P. illuminata, P. japonica, P. kunmingensis, P. metallifera,P. parataibaiensis, P. rufipes, P. semiannulata, P. viridicornuta),Perillus (P. bioculatus, P. confluens, P. strigipes), Picromerus (P.griseus), Piezodorus (P. degeeri, P. guildinii, P. lituratus (gorseshield bug)), Pinthaeus (P. humeralis), Plautia (P. crossota, P. stali(brown-winged green bug)), Podisus (P. maculiventris), Priassus (P.testaceus), Prionosoma, Proxys (P. albopunctulatus, P. punctulatus, P.victor), Rhaphigaster (R. nebulosa), Scotinophara (S. horvathi),Stiretrus (S. anchorago, S. fimbriatus), Thyanta (T. accerra, T.calceata, T. casta, T. perditor, T. pseudocasta), Trichopepla (T.aurora, T. dubia, T. pilipes, T. semivittata, T. vandykei), Tylospilus,and Zicrona.

II. Target Sequences

As used herein, a “target sequence” or “target polynucleotide” comprisesany sequence in the pest that one desires to reduce the level ofexpression. In specific embodiments, decreasing the level of the targetsequence in the pest controls the pest. For instance, the targetsequence can be essential for growth and development. While the targetsequence can be expressed in any tissue of the pest, in specificembodiments, the sequences targeted for suppression in the pest areexpressed in cells of the gut tissue of the pest, cells in the midgut ofthe pest, and cells lining the gut lumen or the midgut. Such targetsequences can be involved in, for example, gut cell metabolism, growthor differentiation. Non-limiting examples of target sequences of theinvention include a polynucleotide set forth in SEQ ID NOS: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278,279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,302, 303, or 304. As exemplified elsewhere herein, decreasing the levelof expression of one or more of these target sequences in a Pentatomidaeplant pest or a N. viridula, Acrosternum hilare, Piezodorus guildini,and/or Halymorpha halys plant pest controls the pest.

III. Silencing Elements

By “silencing element” is intended a polynucleotide which when ingestedby a pest, is capable of reducing or eliminating the level or expressionof a target polynucleotide or the polypeptide encoded thereby. Thesilencing element employed can reduce or eliminate the expression levelof the target sequence by influencing the level of the target RNAtranscript or, alternatively, by influencing translation and therebyaffecting the level of the encoded polypeptide. Methods to assay forfunctional silencing elements that are capable of reducing oreliminating the level of a sequence of interest are disclosed elsewhereherein. A single polynucleotide employed in the methods of the inventioncan comprise one or more silencing elements to the same or differenttarget polynucleotides. The silencing element can be produced in vivo(i.e., in a host cell such as a plant or microorganism) or in vitro.

In specific embodiments, the target sequence is not endogenous to theplant. In other embodiments, while the silencing element controls pests,preferably the silencing element has no effect on the normal plant orplant part.

As discussed in further detail below, silencing elements can include,but are not limited to, a sense suppression element, an antisensesuppression element, a double stranded RNA, a siRNA, an amiRNA, a miRNA,or a hairpin suppression element. Non-limiting examples of silencingelements that can be employed to decrease expression of these targetPentatomidae plant pest sequences or N. viridula, Acrosternum hilare,Piezodorus guildini, and/or Halymorpha halys plant pest sequencescomprise fragments and variants of the sense or antisense sequence orconsists of the sense or antisense sequence of the sequence set forth inSEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, 292, 302, 303, or 304 or a biologically activevariant or fragment thereof. Additional sequences that can be employedas silencing elements include, for example, SEQ ID NOS: 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 305, 306, 307, 308, 309, 310, 311, 312, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, or 336 or activevariants or fragments thereof. The silencing element can furthercomprise additional sequences that advantageously effect transcriptionand/or the stability of a resulting transcript. For example, thesilencing elements can comprise at least one thymine residue at the 3′end. This can aid in stabilization. Thus, the silencing elements canhave at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more thymine residues atthe 3′ end. As discussed in further detail below, enhancer suppressorelements can also be employed in conjunction with the silencing elementsdisclosed herein.

By “reduces” or “reducing” the expression level of a polynucleotide or apolypeptide encoded thereby is intended to mean, the polynucleotide orpolypeptide level of the target sequence is statistically lower than thepolynucleotide level or polypeptide level of the same target sequence inan appropriate control pest which is not exposed to (i.e., has notingested) the silencing element. In particular embodiments of theinvention, reducing the polynucleotide level and/or the polypeptidelevel of the target sequence in a pest according to the inventionresults in less than 95%, less than 90%, less than 80%, less than 70%,less than 60%, less than 50%, less than 40%, less than 30%, less than20%, less than 10%, or less than 5% of the polynucleotide level, or thelevel of the polypeptide encoded thereby, of the same target sequence inan appropriate control pest. Methods to assay for the level of the RNAtranscript, the level of the encoded polypeptide, or the activity of thepolynucleotide or polypeptide are discussed elsewhere herein.

i. Sense Suppression Elements

As used herein, a “sense suppression element” comprises a polynucleotidedesigned to express an RNA molecule corresponding to at least a part ofa target messenger RNA in the “sense” orientation. Expression of the RNAmolecule comprising the sense suppression element reduces or eliminatesthe level of the target polynucleotide or the polypeptide encodedthereby. The polynucleotide comprising the sense suppression element maycorrespond to all or part of the sequence of the target polynucleotide,all or part of the 5′ and/or 3′ untranslated region of the targetpolynucleotide, all or part of the coding sequence of the targetpolynucleotide, or all or part of both the coding sequence and theuntranslated regions of the target polynucleotide.

Typically, a sense suppression element has substantial sequence identityto the target polynucleotide, typically greater than about 65% sequenceidentity, greater than about 85% sequence identity, about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. See, U.S. Pat.Nos. 5,283,184 and 5,034,323; herein incorporated by reference. Thesense suppression element can be any length so long as it allows for thesuppression of the targeted sequence. The sense suppression element canbe, for example, 15, 16, 17, 18 19, 20, 22, 25, 30, 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 600, 700, 900, 1000, 1100, 1200, 1300nucleotides or longer of the target polynucleotides set forth in any ofSEQ ID NO:1-292 or 302-304. In other embodiments, the sense suppressionelement can be, for example, about 15-25, 25-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650,650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,1500-1600, 1600-1700, 1700-1800 nucleotides or longer of the targetpolynucleotides set forth in any of SEQ ID NO: 1-292 or 302-304.

ii. Antisense Suppression Elements

As used herein, an “antisense suppression element” comprises apolynucleotide which is designed to express an RNA moleculecomplementary to all or part of a target messenger RNA. Expression ofthe antisense RNA suppression element reduces or eliminates the level ofthe target polynucleotide. The polynucleotide for use in antisensesuppression may correspond to all or part of the complement of thesequence encoding the target polynucleotide, all or part of thecomplement of the 5′ and/or 3′ untranslated region of the targetpolynucleotide, all or part of the complement of the coding sequence ofthe target polynucleotide, or all or part of the complement of both thecoding sequence and the untranslated regions of the targetpolynucleotide. In addition, the antisense suppression element may befully complementary (i.e., 100% identical to the complement of thetarget sequence) or partially complementary (i.e., less than 100%identical to the complement of the target sequence) to the targetpolynucleotide. In specific embodiments, the antisense suppressionelement comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence complementarity to the target polynucleotide.Antisense suppression may be used to inhibit the expression of multipleproteins in the same plant. See, for example, U.S. Pat. No. 5,942,657.Furthermore, the antisense suppression element can be complementary to aportion of the target polynucleotide. Generally, sequences of at least15, 20, 22, 25, 50, 100, 200, 300, 400, 450 nucleotides or greater ofthe sequence set forth in any of SEQ ID NO: 1-292 or 302-304 may beused. Methods for using antisense suppression to inhibit the expressionof endogenous genes in plants are described, for example, in Liu et al(2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and5,942,657, each of which is herein incorporated by reference.

iii. Double Stranded RNA Suppression Element

A “double stranded RNA silencing element” or “dsRNA” comprises at leastone transcript that is capable of forming a dsRNA either before or afteringestion by a pest. Thus, a “dsRNA silencing element” includes a dsRNA,a transcript or polyribonucleotide capable of forming a dsRNA or morethan one transcript or polyribonucleotide capable of forming a dsRNA.“Double stranded RNA” or “dsRNA” refers to a polyribonucleotidestructure formed either by a single self-complementary RNA molecule or apolyribonucleotide structure formed by the expression of least twodistinct RNA strands. The dsRNA molecule(s) employed in the methods andcompositions of the invention mediate the reduction of expression of atarget sequence, for example, by mediating RNA interference “RNAi” orgene silencing in a sequence-specific manner. In the context of thepresent invention, the dsRNA is capable of reducing or eliminating thelevel or expression of a target polynucleotide or the polypeptideencoded thereby in a pest.

The dsRNA can reduce or eliminate the expression level of the targetsequence by influencing the level of the target RNA transcript, byinfluencing translation and thereby affecting the level of the encodedpolypeptide, or by influencing expression at the pre-transcriptionallevel (i.e., via the modulation of chromatin structure, methylationpattern, etc., to alter gene expression). See, for example, Verdel etal. (2004) Science 303:672-676; Pal-Bhadra et al. (2004) Science303:669-672; Allshire (2002) Science 297:1818-1819; Volpe et al. (2002)Science 297:1833-1837; Jenuwein (2002) Science 297:2215-2218; and Hallet al. (2002) Science 297:2232-2237. Methods to assay for functionaldsRNA that are capable of reducing or eliminating the level of asequence of interest are disclosed elsewhere herein. Accordingly, asused herein, the term “dsRNA” is meant to encompass other terms used todescribe nucleic acid molecules that are capable of mediating RNAinterference or gene silencing, including, for example,short-interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA(miRNA), hairpin RNA, short hairpin RNA (shRNA), post-transcriptionalgene silencing RNA (ptgsRNA), and others.

In specific embodiments, at least one strand of the duplex ordouble-stranded region of the dsRNA shares sufficient sequence identityor sequence complementarity to the target polynucleotide to allow forthe dsRNA to reduce the level of expression of the target sequence. Asused herein, the strand that is complementary to the targetpolynucleotide is the “antisense strand” and the strand homologous tothe target polynucleotide is the “sense strand.”

In another embodiment, the dsRNA comprises a hairpin RNA. A hairpin RNAcomprises an RNA molecule that is capable of folding back onto itself toform a double stranded structure. Multiple structures can be employed ashairpin elements. In specific embodiments, the dsRNA suppression elementcomprises a hairpin element which comprises in the following order, afirst segment, a second segment, and a third segment, where the firstand the third segment share sufficient complementarity to allow thetranscribed RNA to form a double-stranded stem-loop structure.

The “second segment” of the hairpin comprises a “loop” or a “loopregion.” These terms are used synonymously herein and are to beconstrued broadly to comprise any nucleotide sequence that confersenough flexibility to allow self-pairing to occur between complementaryregions of a polynucleotide (i.e., segments 1 and 3 which form the stemof the hairpin). For example, in some embodiments, the loop region maybe substantially single stranded and act as a spacer between theself-complementary regions of the hairpin stem-loop. In someembodiments, the loop region can comprise a random or nonsensenucleotide sequence and thus not share sequence identity to a targetpolynucleotide. In other embodiments, the loop region comprises a senseor an antisense RNA sequence or fragment thereof that shares identity toa target polynucleotide. See, for example, International PatentPublication No. WO 02/00904, herein incorporated by reference. Inspecific embodiments, the loop region can be optimized to be as short aspossible while still providing enough intramolecular flexibility toallow the formation of the base-paired stem region. Accordingly, theloop sequence is generally less than 1000, 900, 800, 700, 600, 500, 400,300, 200, 100, 50, 25, 20, 15, 10 nucleotides or less.

The “first” and the “third” segment of the hairpin RNA molecule comprisethe base-paired stem of the hairpin structure. The first and the thirdsegments are inverted repeats of one another and share sufficientcomplementarity to allow the formation of the base-paired stem region.In specific embodiments, the first and the third segments are fullycomplementary to one another. Alternatively, the first and the thirdsegment may be partially complementary to each other so long as they arecapable of hybridizing to one another to form a base-paired stem region.The amount of complementarity between the first and the third segmentcan be calculated as a percentage of the entire segment. Thus, the firstand the third segment of the hairpin RNA generally share at least 50%,60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, upto and including 100% complementarity.

The first and the third segment are at least about 1000, 500, 400, 300,200, 100, 50, 40, 30, 25, 22, 20, 19, 18, 17, 16, 15 or 10 nucleotidesin length. In specific embodiments, the length of the first and/or thethird segment is about 10-100 nucleotides, about 10 to about 75nucleotides, about 10 to about 50 nucleotides, about 10 to about 40nucleotides, about 10 to about 35 nucleotides, about 10 to about 30nucleotides, about 10 to about 25 nucleotides, about 10 to about 19nucleotides, about 50 nucleotides to about 100 nucleotides, about 100nucleotides to about 150 nucleotides, about 150 nucleotides to about 200nucleotides, about 200 nucleotides to about 250 nucleotides, about 250nucleotides to about 300 nucleotides, about 300 nucleotides to about 350nucleotides, about 350 nucleotides to about 400 nucleotides, about 400nucleotide to about 500 nucleotides, about 600 nt, about 700 nt, about800 nt, about 900 nt, about 1000 nt, about 1100 nt, about 1200 nt, 1300nt, 1400 nt, 1500 nt, 1600 nt, 1700 nt, 1800 nt, 1900 nt, 2000 nt orlonger. In other embodiments, the length of the first and/or the thirdsegment comprises at least 10-19 nucleotides; 19-35 nucleotides; 30-45nucleotides; 40-50 nucleotides; 50-100 nucleotides; 100-300 nucleotides;about 500 -700 nucleotides; about 700-900 nucleotides; about 900-1100nucleotides; about 1300 -1500 nucleotides; about 1500 -1700 nucleotides;about 1700 -1900 nucleotides; about 1900 -2100 nucleotides; about 2100-2300 nucleotides; or about 2300 -2500 nucleotides. See, for example,International Publication No. WO 0200904. In specific embodiments, thefirst and the third segment comprise at least 19 nucleotides having atleast 85% complementary to the first segment. In still otherembodiments, the first and the third segments which form the stem-loopstructure of the hairpin comprises 3′ or 5′ overhang regions havingunpaired nucleotide residues.

In specific embodiments, the sequences used in the first, the second,and/or the third segments comprise domains that are designed to havesufficient sequence identity to a target polynucleotide of interest andthereby have the ability to decrease the level of expression of thetarget polynucleotide. The specificity of the inhibitory RNA transcriptsis therefore generally conferred by these domains of the silencingelement. Thus, in some embodiments of the invention, the first, secondand/or third segment of the silencing element comprise a domain havingat least 10, at least 15, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 30, at least40, at least 50, at least 100, at least 200, at least 300, at least 500,at least 1000, or more than 1000 nucleotides that share sufficientsequence identity to the target polynucleotide to allow for a decreasein expression levels of the target polynucleotide when expressed in anappropriate cell. In other embodiments, the domain is between about 15to 50 nucleotides, about 19-35 nucleotides, about 25-50 nucleotides,about 19 to 75 nucleotides, about 40-90 nucleotides about 15-100nucleotidesl0-100 nucleotides, about 10 to about 75 nucleotides, about10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10to about 35 nucleotides, about 10 to about 30 nucleotides, about 10 toabout 25 nucleotides, about 10 to about 19 nucleotides, about 50nucleotides to about 100 nucleotides, about 100 nucleotides to about 150nucleotides, about 150 nucleotides to about 200 nucleotides, about 200nucleotides to about 250 nucleotides, about 250 nucleotides to about 300nucleotides, about 300 nucleotides to about 350 nucleotides, about 350nucleotides to about 400 nucleotides, about 400 nucleotide to about 500nucleotides or longer. In other embodiments, the length of the firstand/or the third segment comprises at least 10-19 nucleotides, 19-35nucleotides, 30-45 nucleotides, 40-50 nucleotides, 50-100 nucleotides,or about 100-300 nucleotides.

In specific embodiments, the domain of the first, the second, and/or thethird segment has 100% sequence identity to the target polynucleotide.In other embodiments, the domain of the first, the second and/or thethird segment having homology to the target polypeptide have at least50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or greater sequence identity to a region of the targetpolynucleotide. The sequence identity of the domains of the first, thesecond and/or the third segments to the target polynucleotide need onlybe sufficient to decrease expression of the target polynucleotide ofinterest. See, for example, Chuang and Meyerowitz (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk et al. (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38;Pandolfini et al. BMC Biotechnology 3:7, and U.S. Patent Publication No.20030175965; each of which is herein incorporated by reference. Atransient assay for the efficiency of hpRNA constructs to silence geneexpression in vivo has been described by Panstruga et al. (2003) Mol.Biol. Rep. 30:135-140, herein incorporated by reference.

The amount of complementarity shared between the first, second, and/orthird segment and the target polynucleotide or the amount ofcomplementarity shared between the first segment and the third segment(i.e., the stem of the hairpin structure) may vary depending on theorganism in which gene expression is to be controlled. Some organisms orcell types may require exact pairing or 100% identity, while otherorganisms or cell types may tolerate some mismatching. In some cells,for example, a single nucleotide mismatch in the targeting sequenceabrogates the ability to suppress gene expression. In these cells, thesuppression cassettes of the invention can be used to target thesuppression of mutant genes, for example, oncogenes whose transcriptscomprise point mutations and therefore they can be specifically targetedusing the methods and compositions of the invention without altering theexpression of the remaining wild-type allele.

Any region of the target polynucleotide can be used to design the domainof the silencing element that shares sufficient sequence identity toallow expression of the hairpin transcript to decrease the level of thetarget polynucleotide. For instance, the domain can be designed to sharesequence identity to the 5′ untranslated region of the targetpolynucleotide(s), the 3′ untranslated region of the targetpolynucleotide(s), exonic regions of the target polynucleotide(s),intronic regions of the target polynucleotide(s), and any combinationthereof. In specific embodiments, a domain of the silencing elementshares sufficient homology to at least about 15, 16, 17, 18, 19, 20, 22,25 or 30 consecutive nucleotides from about nucleotides 1-50, 25-75,75-125, 50-100, 125-175, 175-225, 100-150, 150-200, 200-250, 225-275,275-325, 250-300, 325-375, 375-425, 300-350, 350-400, 425-475, 400-450,475-525, 450-500, 525-575, 575-625, 550-600, 625-675, 675-725, 600-650,625-675, 675-725, 650-700, 725-825, 825-875, 750-800, 875-925, 925-975,850-900, 925-975, 975-1025, 950-1000, 1000-1050, 1025-1075, 1075-1125,1050-1100, 1125-1175, 1100-1200, 1175-1225, 1225-1275, 1200-1300,1325-1375, 1375-1425, 1300-1400, 1425-1475, 1475-1525, 1400-1500,1525-1575, 1575-1625, 1625-1675, 1675-1725, 1725-1775, 1775-1825,1825-1875, 1875-1925, 1925-1975, 1975-2025, 2025-2075, 2075-2125,2125-2175, 2175-2225, 1500-1600, 1600-1700, 1700-1800, 1800-1900,1900-2000 of the target sequence. In some instances to optimize thesiRNA sequences employed in the hairpin, the syntheticoligodeoxyribonucleotide/RNAse H method can be used to determine siteson the target mRNA that are in a conformation that is susceptible to RNAsilencing. See, for example, Vickers et al. (2003) J. Biol. Chem278:7108-7118 and Yang et al. (2002) Proc. Natl. Acad. Sci. USA99:9442-9447, herein incorporated by reference. These studies indicatethat there is a significant correlation between the RNase-H-sensitivesites and sites that promote efficient siRNA-directed mRNA degradation.

The hairpin silencing element may also be designed such that the sensesequence or the antisense sequence do not correspond to a targetpolynucleotide. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the target polynucleotide. Thus, it is the loop regionthat determines the specificity of the RNA interference. See, forexample, WO 02/00904, herein incorporated by reference.

In addition, transcriptional gene silencing (TGS) may be accomplishedthrough use of a hairpin suppression element where the inverted repeatof the hairpin shares sequence identity with the promoter region of atarget polynucleotide to be silenced. See, for example, Aufsatz et al.(2002) PNAS 99 (Suppl. 4):16499-16506 and Mette et al. (2000) EMBO J19(19):5194-5201.

While the various target sequences disclosed herein can be used todesign any silencing element that encodes a hairpin suppressionconstruct, non-limiting examples of such hairpin constructs are setforth in SEQ ID NO: 293 which targets SEQ ID NO: 278;

SEQ ID NOS: 294, 295 and 296 which target SEQ ID NO: 279; SEQ ID NOS:297 and 298 which target SEQ ID NO:280; SEQ ID NO:299 which targets SEQID NO:281; SEQ ID NO: 300 which targets SEQ ID NO: 282; and SEQ ID NO:301 which targets SEQ ID NO: 283; or active variants or fragmentsthereof. In other embodiments, the dsRNA can comprise a small RNA(sRNA). sRNAs can comprise both micro RNA (miRNA) and short-interferingRNA (siRNA) (Meister and Tuschl (2004) Nature 431:343-349 and Bonetta etal. (2004) Nature Methods 1:79-86). “MicroRNAs” or “miRNAs” areregulatory agents comprising about 19 to about 24 nucleotides (nt) inlength, which are highly efficient at inhibiting the expression oftarget polynucleotides. See, for example Javier et al. (2003) Nature425: 257-263, herein incorporated by reference. For miRNA interference,the silencing element can be designed to express a dsRNA molecule thatforms a hairpin structure containing a 21 nucleotide sequence that iscomplementary to the target polynucleotide of interest. The miRNA can bean “artificial miRNA” or “amiRNA” which comprises a miRNA sequence thatis synthetically designed to silence a target sequence.

When expressing an miRNA, the final (mature) miRNA is present in aduplex in a precursor backbone structure, the two strands being referredto as the miRNA (the strand that will eventually basepair with thetarget) and miRNA* (star sequence). This final miRNA is a substrate fora form of dicer that removes the miRNA/miRNA* duplex from the precursor,after which, similarly to siRNAs, the duplex can be taken into the RISCcomplex. It has been demonstrated that miRNAs can be transgenicallyexpressed and be effective through expression of a precursor form,rather than the entire primary form (Parizotto et al. (2004) Genes &Development 18 :2237-2242 and Guo et al. (2005) Plant Cell17:1376-1386).

The silencing element for miRNA interference comprises a miRNA precursorbackbone. The miRNA precursor backbone comprises a DNA sequence havingthe miRNA and star sequences. When expressed as an RNA, the structure ofthe miRNA precursor backbone is such as to allow for the formation of ahairpin RNA structure that can be processed into a miRNA. In someembodiments, the miRNA precursor backbone comprises a genomic miRNAprecursor sequence, wherein said sequence comprises a native precursorin which an heterologous (artificial) miRNA and star sequence areinserted.

As used herein, a “star sequence” is the sequence within a miRNAprecursor backbone that is complementary to the miRNA and forms a duplexwith the miRNA to form the stem structure of a hairpin RNA. In someembodiments, the star sequence can comprise less than 100%complementarity to the miRNA sequence. Alternatively, the star sequencecan comprise at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% or lowersequence complementarity to the miRNA sequence as long as the starsequence has sufficient complementarity to the miRNA sequence to form adouble stranded structure. In still further embodiments, the starsequence comprises a sequence having 1, 2, 3, 4, 5 or more mismatcheswith the miRNA sequence and still has sufficient complementarity to forma double stranded structure with the miRNA sequence resulting inproduction of miRNA and suppression of the target sequence.

The miRNA precursor backbones can be from any plant. In someembodiments, the miRNA precursor backbone is from a monocot. In otherembodiments, the miRNA precursor backbone is from a dicot. In furtherembodiments, the backbone is from maize or soybean. MicroRNA precursorbackbones have been described previously. For example, US20090155910A1(WO 2009/079532) discloses the following soybean miRNA precursorbackbones: 156c, 159, 166b, 168c, 396b and 398b, and US20090155909A1 (WO2009/079548) discloses the following maize miRNA precursor backbones:159c, 164h, 168a, 169r, and 396h. Each of these references isincorporated by reference in their entirety.

Thus, the miRNA precursor backbone can be altered to allow for efficientinsertion of heterologous miRNA and star sequences within the miRNAprecursor backbone. In such instances, the miRNA segment and the starsegment of the miRNA precursor backbone are replaced with theheterologous miRNA and the heterologous star sequences, designed totarget any sequence of interest, using a PCR technique and cloned intoan expression construct. It is recognized that there could bealterations to the position at which the artificial miRNA and starsequences are inserted into the backbone. Detailed methods for insertingthe miRNA and star sequence into the miRNA precursor backbone aredescribed elsewhere herein (see, Example 8) and are also described in,for example, US Patent Applications 20090155909A1 and US20090155910A1,herein incorporated by reference in their entirety.

When designing a miRNA sequence and star sequence, various designchoices can be made. See, for example, Schwab R, et al. (2005) Dev Cell8: 517-27. In non-limiting embodiments, the miRNA sequences disclosedherein can have a “U” at the 5′-end, a “C” or “G” at the 19^(th)nucleotide position, and an “A” or “U” at the 10th nucleotide position.In other embodiments, the miRNA design is such that the miRNA have ahigh free delta-G as calculated using the ZipFold algorithm (Markham, N.R. & Zuker, M. (2005) Nucleic Acids Res. 33: W577-W581.) Optionally, aone base pair change can be added within the 5′ portion of the miRNA sothat the sequence differs from the target sequence by one nucleotide.

The methods and compositions of the invention employ silencing elementsthat when transcribed “form” a dsRNA molecule. Accordingly, theheterologous polynucleotide being expressed need not form the dsRNA byitself, but can interact with other sequences in the plant cell or inthe pest gut after ingestion to allow the formation of the dsRNA. Forexample, a chimeric polynucleotide that can selectively silence thetarget polynucleotide can be generated by expressing a chimericconstruct comprising the target sequence for a miRNA or siRNA to asequence corresponding to all or part of the gene or genes to besilenced. In this embodiment, the dsRNA is “formed” when the target forthe miRNA or siRNA interacts with the miRNA present in the cell. Theresulting dsRNA can then reduce the level of expression of the gene orgenes to be silenced. See, for example, US Application Publication2007-0130653, entitled “Methods and Compositions for Gene Silencing”,herein incorporated by reference. The construct can be designed to havea target for an endogenous miRNA or alternatively, a target for aheterologous and/or synthetic miRNA can be employed in the construct. Ifa heterologous and/or synthetic miRNA is employed, it can be introducedinto the cell on the same nucleotide construct as the chimericpolynucleotide or on a separate construct. As discussed elsewhereherein, any method can be used to introduce the construct comprising theheterologous miRNA.

While the various target sequences disclosed herein can be used todesign any silencing element that encodes a miRNA, non-limiting examplesof such miRNA constructs include SEQ ID NOS: 311, 312, 327, 328, 335 or336 which target SEQ ID NO: 304; SEQ ID NOS: 307, 308, 323, 324, 331 or332 which target SEQ ID NO: 278; SEQ ID NOS: 309, 310, 325, 326, 333 or334 which target SEQ ID NO: 303; and SEQ ID NOS: 305, 306, 321, 322, 329or 330 which target SEQ ID NO: 302; or active variants or fragmentsthereof.

IV. Variants and Fragments

By “fragment” is intended a portion of the polynucleotide or a portionof the amino acid sequence and hence protein encoded thereby. Fragmentsof a polynucleotide may encode protein fragments that retain thebiological activity of the native protein. Alternatively, fragments of apolynucleotide that are useful as a silencing element do not need toencode fragment proteins that retain biological activity. Thus,fragments of a nucleotide sequence may range from at least about 10,about 15, about 16, about 17, about 18, about 19, about 20 nucleotides,about 22 nucleotides, about 50 nucleotides, about 75 nucleotides, about100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500nucleotides, 600 nucleotides, 700 nucleotides and up to the full-lengthpolynucleotide employed in the invention. Alternatively, fragments of anucleotide sequence may range from 1-50, 25-75, 75-125, 50-100, 125-175,175-225, 100-150, 150-200, 200-250, 225-275, 275-325, 250-300, 325-375,375-425, 300-350, 350-400, 425-475, 400-450, 475-525, 450-500, 525-575,575-625, 550-600, 625-675, 675-725, 600-650, 625-675, 675-725, 650-700,725-825, 825-875, 750-800, 875-925, 925-975, 850-900, 925-975, 975-1025,950-1000, 1000-1050, 1025-1075, 1075-1125, 1050-1100, 1125-1175,1100-1200, 1175-1225, 1225-1275, 1200-1300, 1325-1375, 1375-1425,1300-1400, 1425-1475, 1475-1525, 1400-1500, 1525-1575, 1575-1625,1625-1675, 1675-1725, 1725-1775, 1775-1825, 1825-1875, 1875-1925,1925-1975, 1975-2025, 2025-2075, 2075-2125, 2125-2175, 2175-2225,1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000 of any one of SEQID NOS: 1-304 or 321-336. Methods to assay for the activity of a desiredsilencing element are described elsewhere herein.

Encompassed herein are fragments of the various target sequences (i.e.SEQ ID NOS: 1-292 and 302-304) which are useful as silencing elementsand fragments of the various silencing elements provided herein (i.e.SEQ ID NOS:293-301 or 321-336). Thus, fragments of a nucleotide sequencethat are useful as silencing elements may range from at least about 10,about 15, about 16, about 17, about 18, about 19, about 20 nucleotides,about 22 nucleotides, about 50 nucleotides, about 75 nucleotides, about100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500nucleotides, 600 nucleotides, 700 nucleotides and up to the full-lengthpolynucleotide sequences of SEQ ID NOS: 1-304 or 321-336. Alternatively,fragments of a nucleotide sequence that are useful as silencing elementsmay range from 1-50, 25-75, 75-125, 50-100, 125-175, 175-225, 100-150,150-200, 200-250, 225-275, 275-325, 250-300, 325-375, 375-425, 300-350,350-400, 425-475, 400-450, 475-525, 450-500, 525-575, 575-625, 550-600,625-675, 675-725, 600-650, 625-675, 675-725, 650-700, 725-825, 825-875,750-800, 875-925, 925-975, 850-900, 925-975, 975-1025, 950-1000,1000-1050, 1025-1075, 1075-1125, 1050-1100, 1125-1175, 1100-1200,1175-1225, 1225-1275, 1200-1300, 1325-1375, 1375-1425, 1300-1400,1425-1475, 1475-1525, 1400-1500, 1525-1575, 1575-1625, 1625-1675,1675-1725, 1725-1775, 1775-1825, 1825-1875, 1875-1925, 1925-1975,1975-2025, 2025-2075, 2075-2125, 2125-2175, 2175-2225, 1500-1600,1600-1700, 1700-1800, 1800-1900, 1900-2000 of any one of SEQ ID NOS:1-304 or 321-336. Methods to assay for the activity of a desiredsilencing element are described elsewhere herein. Various, non-limitingexamples of fragments of SEQ ID NOS: 1-292 or 302-304 are providedherein and include, for example, SEQ ID NOS: 284-292 or 305-312.

“Variants” is intended to mean substantially similar sequences. Thus,further provided are variants of the various sequences set forth in SEQID NOS: 1-336. For polynucleotides, a variant comprises a deletionand/or addition of one or more nucleotides at one or more internal siteswithin the native polynucleotide and/or a substitution of one or morenucleotides at one or more sites in the native polynucleotide. A variantof a polynucleotide that is useful as a silencing element will retainthe ability to reduce expression of the target polynucleotide and, insome embodiments, thereby control a pest of interest. As used herein, a“native” polynucleotide or polypeptide comprises a naturally occurringnucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the polypeptides employed in the invention. Variantpolynucleotides also include synthetically derived polynucleotide, suchas those generated, for example, by using site-directed mutagenesis, butcontinue to retain the desired activity. Generally, variants of aparticular polynucleotide of the invention (i.e., a silencing element)will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to that particular polynucleotide as determined by sequencealignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Percent sequence identity between any two polypeptidescan be calculated using sequence alignment programs and parametersdescribed elsewhere herein. Where any given pair of polynucleotidesemployed in the invention is evaluated by comparison of the percentsequence identity shared by the two polypeptides they encode, thepercent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, California).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

A method is further provided for identifying a silencing element fromthe target polynucleotides set froth in SEQ ID NO: 1-292 or 302-304.Such methods comprise obtaining a candidate fragment of any one of SEQID NO: 1-292 or 302-304 which is of sufficient length to act as asilencing element and thereby reduce the expression of the targetpolynucleotide and/or control a desired pest; expressing said candidatepolynucleotide fragment in an appropriate expression cassette to producea candidate silencing element and determining is said candidatepolynucleotide fragment has the activity of a silencing element andthereby reduce the expression of the target polynucleotide and/orcontrols a desired pest. Methods of identifying such candidate fragmentsbased on the desired pathway for suppression are known. For example,various bioinformatics programs can be employed to identify the regionof the target polynucleotides that could be exploited to generate asilencing element. See, for example, Elbahir et al. (2001) Genes andDevelopment 15:188-200, Schwartz et al. (2003) Cell 115:199-208,Khvorova et al. (2003) Cell 115:209-216.

See also, siRNA at Whitehead (jura.wi.mit.edu/bioc/siRNAext/) whichcalculates the binding energies for both sense and antisense siRNAs.See, also genscript.com/ssl-bin/app/rnai?op=known; Block-iT™ RNAidesigner from Invitrogen and GenScript siRNA Construct Builder.

V. DNA Constructs

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

The polynucleotide encoding the silencing element or in specificembodiments employed in the methods and compositions of the inventioncan be provided in expression cassettes for expression in a plant ororganism of interest. It is recognized that multiple silencing elementsincluding multiple identical silencing elements, multiple silencingelements targeting different regions of the target sequence, or multiplesilencing elements from different target sequences can be used. In thisembodiment, it is recognized that each silencing element can becontained in a single or separate cassette, DNA construct, or vector. Asdiscussed, any means of providing the silencing element is contemplated.A plant or plant cell can be transformed with a single cassettecomprising DNA encoding one or more silencing elements or separatecassettes comprising each silencing element can be used to transform aplant or plant cell or host cell. Likewise, a plant transformed with onecomponent can be subsequently transformed with the second component. Oneor more silencing elements can also be brought together by sexualcrossing. That is, a first plant comprising one component is crossedwith a second plant comprising the second component. Progeny plants fromthe cross will comprise both components.

The expression cassette can include 5′ and 3′ regulatory sequencesoperably linked to the polynucleotide of the invention. “Operablylinked” is intended to mean a functional linkage between two or moreelements. For example, an operable linkage between a polynucleotide ofthe invention and a regulatory sequence (i.e., a promoter) is afunctional link that allows for expression of the polynucleotide of theinvention. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame. The cassette may additionally contain at least oneadditional polynucleotide to be cotransformed into the organism.Alternatively, the additional polypeptide(s) can be provided on multipleexpression cassettes. Expression cassettes can be provided with aplurality of restriction sites and/or recombination sites for insertionof the polynucleotide to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a polynucleotide comprising the silencing elementemployed in the methods and compositions of the invention, and atranscriptional and translational termination region (i.e., terminationregion) functional in plants. In other embodiment, the double strandedRNA is expressed from a suppression cassette. Such a cassette cancomprise two convergent promoters that drive transcription of anoperably linked silencing element. “Convergent promoters” refers topromoters that are oriented on either terminus of the operably linkedsilencing element such that each promoter drives transcription of thesilencing element in opposite directions, yielding two transcripts. Insuch embodiments, the convergent promoters allow for the transcriptionof the sense and anti-sense strand and thus allow for the formation of adsRNA.

The regulatory regions (i.e., promoters, transcriptional regulatoryregions, and translational termination regions) and/or thepolynucleotides employed in the invention may be native/analogous to thehost cell or to each other. Alternatively, the regulatory regions and/orthe polynucleotide employed in the invention may be heterologous to thehost cell or to each other. As used herein, “heterologous” in referenceto a sequence is a sequence that originates from a foreign species, or,if from the same species, is substantially modified from its native formin composition and/or genomic locus by deliberate human intervention.For example, a promoter operably linked to a heterologous polynucleotideis from a species different from the species from which thepolynucleotide was derived, or, if from the same/analogous species, oneor both are substantially modified from their original form and/orgenomic locus, or the promoter is not the native promoter for theoperably linked polynucleotide. As used herein, a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked polynucleotide encodingthe silencing element, may be native with the plant host, or may bederived from another source (i.e., foreign or heterologous) to thepromoter, the polynucleotide comprising silencing element, the planthost, or any combination thereof. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acids Res. 15:9627-9639.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepolynucleotide encoding the silencing element can be combined withconstitutive, tissue-preferred, or other promoters for expression inplants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026); soybean elongation factor 1A (ACUP01009998), and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

An inducible promoter, for instance, a pathogen-inducible promoter couldalso be employed. Such promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. PlantPathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and VanLoon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, hereinincorporated by reference.

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced expressionwithin a particular plant tissue. Tissue-preferred promoters includeYamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997)Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

In one embodiment, the various silencing elements disclosed herein areexpressed using a seed-preferred promoter. “Seed-preferred” promotersinclude both “seed-specific” promoters (those promoters active duringseed development such as promoters of seed storage proteins) as well as“seed-germinating” promoters (those promoters active during seedgermination). See Thompson et al. (1989) BioEssays 10:108, hereinincorporated by reference. Such seed-preferred promoters include, butare not limited to, Cim1 (cytokinin-induced message); Kunitz trypsininhibitor 3 (kti3) (Genbank accession AF233296); glycinin-1 genes(Genbank accession AB353075.1); cZ19B1 (maize 19 kDa zein); milps(myo-inositol-1-phosphate synthase) (see WO 00/11177 and U.S. Pat. No.6,225,529; herein incorporated by reference). Gamma-zein is anendosperm-specific promoter. Globulin 1 (Glb-1) is a representativeembryo-specific promoter. For dicots, seed-preferred promoters include,but are not limited to, bean β-phaseolin, napin, β-conglycinin alpha(Genbank accession GU723691), soybean lectin, cruciferin, and the like.For monocots, seed-preferred promoters include, but are not limited to,maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken1, shrunken 2, Globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptll (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

In one embodiment of this invention the plant-expressed promoter is avascular-specific promoter such as a phloem-specific promoter. A“vascular-specific” promoter, as used herein, is a promoter which is atleast expressed in vascular cells, or a promoter which is preferentiallyexpressed in vascular cells. Expression of a vascular-specific promoterneed not be exclusively in vascular cells, expression in other celltypes or tissues is possible. A “phloem-specific promoter” as usedherein, is a plant-expressible promoter which is at least expressed inphloem cells, or a promoter which is preferentially expressed in phloemcells.

Expression of a phloem-specific promoter need not be exclusively inphloem cells, expression in other cell types or tissues, e.g., xylemtissue, is possible. In one embodiment of this invention, aphloem-specific promoter is a plant-expressible promoter at leastexpressed in phloem cells, wherein the expression in non-phloem cells ismore limited (or absent) compared to the expression in phloem cells.Examples of suitable vascular-specific or phloem-specific promoters inaccordance with this invention include but are not limited to thepromoters selected from the group consisting of: the SCSV3, SCSV4,SCSVS, and SCSV7 promoters (Schunmann et al. (2003) Plant FunctionalBiology 30:453-60; the rolC gene promoter of Agrobacterium rhizogenes(Kiyokawa et al. (1994) Plant Physiology 104:801-02; Pandolfini et al.(2003) BioMedCentral (BMC) Biotechnology 3:7,(www.biomedcentral.com/1472-6750/3/7); Graham et al. (1997) Plant Mol.Biol. 33:729-35; Guivarc'h et al. (1996); Almon et al. (1997) PlantPhysiol. 115:1599-607; the rolA gene promoter of Agrobacteriumrhizogenes (Dehio et al. (1993) Plant Mol. Biol. 23:1199-210); thepromoter of the Agrobacterium tumefaciens T-DNA gene 5 (Korber et al.(1991) EMBO J. 10:3983-91); the rice sucrose synthase RSs1 gene promoter(Shi et al. (1994) J. Exp. Bot. 45:623-31); the CoYMV or Commelinayellow mottle badnavirus promoter (Medberry et al. (1992) Plant Cell4:185-92; Zhou et al. (1998) Chin. J. Biotechnol. 14:9-16); the CFDV orcoconut foliar decay virus promoter (Rohde et al. (1994) Plant Mol.Biol. 27:623-28; Hehn and Rhode (1998) J. Gen. Virol. 79:1495-99); theRTBV or rice tungro bacilliform virus promoter (Yin and Beachy (1995)Plant J. 7:969-80; Yin et al. (1997) Plant J. 12:1179-80); the peaglutamin synthase GS3A gene (Edwards et al. (1990) Proc. Natl. Acad.Sci. USA 87:3459-63; Brears et al. (1991) Plant J. 1:235-44); the invCD111 and inv CD141 promoters of the potato invertase genes (Hedley etal. (2000) J. Exp. Botany 51:817-21); the promoter isolated fromArabidopsis shown to have phloem-specific expression in tobacco byKertbundit et al. (1991) Proc. Natl. Acad. Sci. USA 88:5212-16); theVAHOX1 promoter region (Tornero et al. (1996) Plant J. 9:639-48); thepea cell wall invertase gene promoter (Zhang et al. (1996) PlantPhysiol. 112:1111-17); the promoter of the endogenous cotton proteinrelated to chitinase of US published patent application 20030106097, anacid invertase gene promoter from carrot (Ramloch-Lorenz et al. (1993)The Plant J. 4:545-54); the promoter of the sulfate transportergeneSultrl; 3 (Yoshimoto et al. (2003) Plant Physiol. 131:1511-17); apromoter of a sucrose synthase gene (Nolte and Koch (1993) PlantPhysiol. 101:899-905); and the promoter of a tobacco sucrose transportergene (Kuhn et al. (1997) Science 275-1298-1300).

Possible promoters also include the Black Cherry promoter for PrunasinHydrolase (PH DL1.4 PRO) (U.S. Pat. No. 6,797, 859), Thioredoxin Hpromoter from cucumber and rice (Fukuda A et al. (2005). Plant CellPhysiol. 46(11):1779-86), Rice (RSs1) (Shi, T. Wang et al. (1994). J.Exp. Bot. 45(274): 623-631) and maize sucrose synthese −1 promoters(Yang., N-S. et al. (1990) PNAS 87:4144-4148), PP2 promoter from pumpkinGuo, H. et al. (2004) Transgenic Research 13:559-566), At SUC2 promoter(Truernit, E. et al. (1995) Planta 196(3):564-70., At SAM-1(S-adenosylmethionine synthetase) (Mijnsbrugge KV. et al. (1996) Planr.Cell. Physiol. 37(8): 1108-1115), and the Rice tungro bacilliform virus(RTBV) promoter (Bhattacharyya-Pakrasi et al. (1993) Plant J.4(1):71-79).

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal.(1988) Cell 52:713-722; Deuschle etal. (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used in the present invention.

VI. Compositions Comprising Silencing Elements

One or more of the polynucleotides comprising the silencing element canbe provided as an external composition such as a spray or powder to theplant, plant part, seed, a pest, or an area of cultivation. In anotherexample, a plant is transformed with a DNA construct or expressioncassette for expression of at least one silencing element. In eithercomposition, the silencing element, when ingested by an insect, canreduce the level of a target pest sequence and thereby control the pest(i.e., a Pentatomidae plant pest including a N. viridula, Acrosternumhilare, Piezodorus guildini, and/or Halymorpha halys. It is recognizedthat the composition can comprise a cell (such as plant cell or abacterial cell), in which a polynucleotide encoding the silencingelement is stably incorporated into the genome and operably linked topromoters active in the cell. Compositions comprising a mixture ofcells, some cells expressing at least one silencing element are alsoencompassed. In other embodiments, compositions comprising the silencingelements are not contained in a cell. In such embodiments, thecomposition can be applied to an area inhabited by a pest. In oneembodiment, the composition is applied externally to a plant (i.e., byspraying a field or area of cultivation) to protect the plant from thepest.

The composition of the invention can further be formulated as bait. Inthis embodiment, the compositions comprise a food substance or anattractant which enhances the attractiveness of the composition to thepest.

The composition comprising the silencing element can be formulated in anagriculturally suitable and/or environmentally acceptable carrier. Suchcarriers can be any material that the animal, plant or environment to betreated can tolerate. Furthermore, the carrier must be such that thecomposition remains effective at controlling a pest. Examples of suchcarriers include water, saline, Ringer's solution, dextrose or othersugar solutions, Hank's solution, and other aqueous physiologicallybalanced salt solutions, phosphate buffer, bicarbonate buffer and Trisbuffer. In addition, the composition may include compounds that increasethe half-life of a composition.

It is recognized that the polynucleotides comprising sequences encodingthe silencing element can be used to transform organisms to provide forhost organism production of these components, and subsequent applicationof the host organism to the environment of the target pest(s). Such hostorganisms include baculoviruses, bacteria, and the like. In this manner,the combination of polynucleotides encoding the silencing element may beintroduced via a suitable vector into a microbial host, and said hostapplied to the environment, or to plants or animals.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may be stablyincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon,or transiently expressed (e.g., transfected mRNA).

Microbial hosts that are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops ofinterest may be selected. These microorganisms are selected so as to becapable of successfully competing in the particular environment with thewild-type microorganisms, provide for stable maintenance and expressionof the sequences encoding the silencing element, and desirably, providefor improved protection of the components from environmental degradationand inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli andAzotobacter vinlandir, and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Of particular interest are the pigmentedmicroorganisms.

A number of ways are available for introducing the polynucleotidecomprising the silencing element into the microbial host underconditions that allow for stable maintenance and expression of suchnucleotide encoding sequences. For example, expression cassettes can beconstructed which include the nucleotide constructs of interest operablylinked with the transcriptional and translational regulatory signals forexpression of the nucleotide constructs, and a nucleotide sequencehomologous with a sequence in the host organism, whereby integrationwill occur, and/or a replication system that is functional in the host,whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include, but arenot limited to, promoters, transcriptional initiation start sites,operators, activators, enhancers, other regulatory elements, ribosomalbinding sites, an initiation codon, termination signals, and the like.See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2;Sambrook et al. (2000); Molecular Cloning: A Laboratory Manual (3rd ed.;Cold Spring Harbor Laboratory Press, Plainview, N.Y.); Davis et al.(1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.); and the references cited therein.

Suitable host cells include the prokaryotes and the lower eukaryotes,such as fungi. Illustrative prokaryotes, both Gram-negative andGram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia,Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such asRhizobium; Spirillaceae, such as photobacterium, Zymomonas , Serratia,Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae;Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceaeand Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetesand Ascomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of the invention include ease of introducing the codingsequence into the host, availability of expression systems, efficiencyof expression, stability in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities, such as thick cell walls,pigmentation, and intracellular packaging or formation of inclusionbodies; leaf affinity; lack of mammalian toxicity; attractiveness topests for ingestion; and the like. Other considerations include ease offormulation and handling, economics, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorulaspp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp.,phylloplane organisms such as Pseudomonas spp., Erwinia spp., andFlavobacterium spp., and other such organisms, including Pseudomonasaeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillusthuringiensis, Escherichia coli, Bacillus subtilis, and the like.

The sequences encoding the silencing elements encompassed by theinvention can be introduced into microorganisms that multiply on plants(epiphytes) to deliver these components to potential target pests.Epiphytes, for example, can be gram-positive or gram-negative bacteria.

The silencing element can be fermented in a bacterial host and theresulting bacteria processed and used as a microbial spray in the samemanner that Bacillus thuringiensis strains have been used asinsecticidal sprays. Any suitable microorganism can be used for thispurpose. Pseudomonas has been used to express Bacillus thuringiensisendotoxins as encapsulated proteins and the resulting cells processedand sprayed as an insecticide Gaertner et al. (1993), in AdvancedEngineered Pesticides, ed. L. Kim (Marcel Decker, Inc.).

Alternatively, the components of the invention are produced byintroducing heterologous genes into a cellular host. Expression of theheterologous sequences results, directly or indirectly, in theintracellular production of the silencing element. These compositionsmay then be formulated in accordance with conventional techniques forapplication to the environment hosting a target pest, e.g., soil, water,and foliage of plants. See, for example, EPA 0192319, and the referencescited therein.

In the present invention, a transformed microorganism can be formulatedwith an acceptable carrier into separate or combined compositions thatare, for example, a suspension, a solution, an emulsion, a dustingpowder, a dispersible granule, a wettable powder, and an emulsifiableconcentrate, an aerosol, an impregnated granule, an adjuvant, a coatablepaste, and also encapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the present invention (i.e., at least one silencingelement) are normally applied in the form of compositions and can beapplied to the crop area, plant, or seed to be treated. For example, thecompositions may be applied to grain in preparation for or duringstorage in a grain bin or silo, etc. The compositions may be appliedsimultaneously or in succession with other compounds. Methods ofapplying an active ingredient or a composition that contains at leastone silencing element include, but are not limited to, foliarapplication, seed coating, and soil application. The number ofapplications and the rate of application depend on the intensity ofinfestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; carboxylate ofa long chain fatty acid; an N-acylsarcosinate; mono- or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitan fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions comprising the silencing element can be in a suitableform for direct application or as a concentrate of primary compositionthat requires dilution with a suitable quantity of water or otherdilutant before application.

The compositions (including the transformed microorganisms) can beapplied to the environment of an insect pest (such as a Pentatomidaeplant pest or a N. viridula, Acrosternum hilare, Piezodorus guildini,and/or Halymorpha halys plant pest) by, for example, spraying,atomizing, dusting, scattering, coating or pouring, introducing into oron the soil, introducing into irrigation water, by seed treatment orgeneral application or dusting at the time when the pest has begun toappear or before the appearance of pests as a protective measure. Forexample, the composition(s) and/or transformed microorganism(s) may bemixed with grain to protect the grain during storage. It is generallyimportant to obtain good control of pests in the early stages of plantgrowth, as this is the time when the plant can be most severely damaged.The compositions can conveniently contain another insecticide if this isthought necessary. In an embodiment of the invention, the composition(s)is applied directly to the soil, at a time of planting, in granular formof a composition of a carrier and dead cells of a Bacillus strain ortransformed microorganism of the invention. Another embodiment is agranular form of a composition comprising an agrochemical such as, forexample, a herbicide, an insecticide, a fertilizer, in an inert carrier,and dead cells of a Bacillus strain or transformed microorganism of theinvention.

VII. Plants, Plant Parts, and Methods of Introducing Sequences intoPlants

The methods of the invention involve introducing a polynucleotide into aplant. In one embodiment, a plant cell is provided having stablyincorporated into its genome a heterologous polynucleotide comprisingany of the various silencing elements provided herein. It is recognizedthat the silencing element, when ingested by a Pentatomidae plant pest,can reduce the level of expression of any of the target sequencesdescrbed herein (i.e. SEQ ID NOS: 1-292 or 302-304). “Introducing” isintended to mean presenting to the plant the polynucleotide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotides into plants are known inthe art including, but not limited to, stable transformation methods,transient transformation methods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. Nos.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and,5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation(WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783;and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize);Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-VanSlogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York),pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the silencing element sequences of theinvention can be provided to a plant using a variety of transienttransformation methods. Such transient transformation methods include,but are not limited to, the introduction of the protein or variants andfragments thereof directly into the plant or the introduction of thetranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway etal. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci.44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107:775-784, all of whichare herein incorporated by reference. Alternatively, polynucleotides canbe transiently transformed into the plant using techniques known in theart. Such techniques include viral vector system and the precipitationof the polynucleotide in a manner that precludes subsequent release ofthe DNA. Thus, the transcription from the particle-bound DNA can occur,but the frequency with which it is released to become integrated intothe genome is greatly reduced. Such methods include the use of particlescoated with polyethylimine (PEI; Sigma #P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. Further, it is recognized that promoters of the invention alsoencompass promoters utilized for transcription by viral RNA polymerases.Methods for introducing polynucleotides into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

As used herein, the term plant also includes plant cells, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants such as embryos, pollen, ovules, seeds,leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks,roots, root tips, anthers, and the like. Grain is intended to mean themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants, and mutants ofthe regenerated plants are also included within the scope of theinvention, provided that these parts comprise the introducedpolynucleotides.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn (Zeamays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularlythose Brassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsand sugarcane plants are optimal, and in yet other embodiments cornplants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

In specific embodiments, the plants/plant cells and/or seeds comprisingan expression construct comprise a silencing element directed to atarget sequence provided herein (i.e. SEQ ID NOS: 1-292 or 302-304)operably linked to a seed-preferred promoter.

VIII. Methods of Use

The methods of the invention comprise methods for controlling a pest(i.e., a Pentatomidae plant pest, such as, N. viridula, Acrosternumhilare, Piezodorus guildini, and/or Halymorpha halys plant pest). Themethod comprises feeding to a pest a composition comprising a silencingelement of the invention, wherein said silencing element, when ingestedby a pest (i.e., a Pentatomidae plant pest including N. viridula,Acrosternum hilare, Piezodorus guildini, and/or Halymorpha halys),reduces the level of a target polynucleotide of the pest and therebycontrols the pest. The pest can be fed the silencing element in avariety of ways. For example, in one embodiment, the polynucleotidecomprising the silencing element is introduced into a plant. As thePentatomidae plant pest or N. viridula, Acrosternum hilare, Piezodorusguildini, and/or Halymorpha halys plant pest feeds on the plant or partthereof expressing these sequences, the silencing element is deliveredto the pest. When the silencing element is delivered to the plant inthis manner, it is recognized that the silencing element can beexpressed constitutively or alternatively, it may be produced in astage-specific manner by employing the various inducible ortissue-preferred or developmentally regulated promoters that arediscussed elsewhere herein. In one embodiment, the silencing element isoperably linked to a seed-preferred promoter. In specific embodiments,the silencing element expressed in the roots, stalk or stem, leafincluding pedicel, xylem and phloem, fruit or reproductive tissue, silk,flowers and all parts therein or any combination thereof.

In another method, a composition comprising at least one silencingelement of the invention is applied to a plant. In such embodiments, thesilencing element can be formulated in an agronomically suitable and/orenvironmentally acceptable carrier, which is preferably, suitable fordispersal in fields. In addition, the carrier can also include compoundsthat increase the half life of the composition. In specific embodiments,the composition comprising the silencing element is formulated in such amanner such that it persists in the environment for a length of timesufficient to allow it to be delivered to a pest. In such embodiments,the composition can be applied to an area inhabited by a pest. In oneembodiment, the composition is applied externally to a plant (i.e., byspraying a field) to protect the plant from pests.

In certain embodiments, the constructs of the present invention can bestacked with any combination of polynucleotide sequences of interest inorder to create plants with a desired trait. A trait, as used herein,refers to the phenotype derived from a particular sequence or groups ofsequences. For example, the polynucleotides of the present invention maybe stacked with any other polynucleotides encoding polypeptides havingpesticidal and/or insecticidal activity, such as other Bacillusthuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986)Gene 48:109), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825,pentin (described in U.S. Pat. No. 5,981,722), and the like. Thecombinations generated can also include multiple copies of any one ofthe polynucleotides of interest. The polynucleotides of the presentinvention can also be stacked with any other gene or combination ofgenes to produce plants with a variety of desired trait combinationsincluding, but not limited to, traits desirable for animal feed such ashigh oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids(e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802;and 5,703,409); barley high lysine (Williamson et al. (1987) Eur. J.Biochem. 165:99-106; and WO 98/20122) and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12:123));increased digestibility (e.g., modified storage proteins (U.S.application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins(U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)); thedisclosures of which are herein incorporated by reference.

The polynucleotides of the present invention can also be stacked withtraits desirable for disease or herbicide resistance (e.g., fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene)); and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils(e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase),starch synthases (SS), starch branching enzymes (SBE), and starchdebranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S.Pat. No. 5.602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs));the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the present invention withpolynucleotides providing agronomic traits such as male sterility (e.g.,see U.S. Pat. No. 5.583,210), stalk strength, flowering time, ortransformation technology traits such as cell cycle regulation or genetargeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); thedisclosures of which are herein incorporated by reference.

These stacked combinations can be created by any method including, butnot limited to, cross-breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Methods and compositions are further provided which allow for anincrease in RNAi produced from the silencing element. In suchembodiments, the methods and compositions employ a first polynucleotidecomprising a silencing element for a target pest sequence operablylinked to a promoter active in the plant cell; and, a secondpolynucleotide comprising a suppressor enhancer element comprising thetarget pest sequence or an active variant or fragment thereof operablylinked to a promoter active in the plant cell. The combined expressionof the silencing element with suppressor enhancer element leads to anincreased amplification of the inhibitory RNA produced from thesilencing element over that achievable with only the expression of thesilencing element alone. In addition to the increased amplification ofthe specific RNAi species itself, the methods and compositions furtherallow for the production of a diverse population of RNAi species thatcan enhance the effectiveness of disrupting target gene expression. Assuch, when the suppressor enhancer element is expressed in a plant cellin combination with the silencing element, the methods and compositioncan allow for the systemic production of RNAi throughout the plant; theproduction of greater amounts of RNAi than would be observed with justthe silencing element construct alone; and, the improved loading of RNAiinto the phloem of the plant, thus providing better control of phloemfeeding insects by an RNAi approach. Thus, the various methods andcompositions provide improved methods for the delivery of inhibitory RNAto the target organism. See, for example, U.S. application Ser. No.12/351,093, entitled “Compositions and Methods for the Suppression ofTarget Polynucleotides”, filed Jan. 9, 2009 and herein incorporated byreference in its entirety.

As used herein, a “suppressor enhancer element” comprises apolynucleotide comprising the target sequence to be suppressed or anactive fragment or variant thereof. It is recognize that the suppressorenhancer element need not be identical to the target sequence, butrather, the suppressor enhancer element can comprise a variant of thetarget sequence, so long as the suppressor enhancer element hassufficient sequence identity to the target sequence to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Similarly,the suppressor enhancer element can comprise a fragment of the targetsequence, wherein the fragment is of sufficient length to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Thus, inspecific embodiments, the suppressor enhancer element comprises apolynucleotide set forth in SEQ ID NO: 1-292, or 302-304 or an activevariant or fragment thereof.

It is recognized that multiple suppressor enhancer elements from thesame target sequence or from different target sequences, or fromdifferent regions of the same target sequence can be employed. Forexample, the suppressor enhancer elements employed can comprisefragments of the target sequence derived from different region of thetarget sequence (i.e., from the 3′UTR, coding sequence, intron, and/or5′UTR). Further, the suppressor enhancer element can be contained in anexpression cassette, as described elsewhere herein, and in specificembodiments, the suppressor enhancer element is on the same or on adifferent DNA vector or construct as the silencing element. Thesuppressor enhancer element can be operably linked to a promoter asdisclosed herein. It is recognized that the suppressor enhancer elementcan be expressed constitutively or alternatively, it may be produced ina stage-specific manner employing the various inducible ortissue-preferred or developmentally regulated promoters that arediscussed elsewhere herein.

In specific embodiments, employing both a silencing element and thesuppressor enhancer element the systemic production of RNAi occursthroughout the entire plant. In further embodiments, the plant or plantparts of the invention have an improved loading of RNAi into the phloemof the plant than would be observed with the expression of the silencingelement construct alone and, thus provide better control of phloemfeeding insects by an RNAi approach. In specific embodiments, theplants, plant parts, and plant cells of the invention can further becharacterized as allowing for the production of a diversity of RNAispecies that can enhance the effectiveness of disrupting target geneexpression.

In specific embodiments, the combined expression of the silencingelement and the suppressor enhancer element increases the concentrationof the inhibitory RNA in the plant cell, plant, plant part, plant tissueor phloem over the level that is achieved when the silencing element isexpressed alone.

As used herein, an “increased level of inhibitory RNA” comprises anystatistically significant increase in the level of RNAi produced in aplant having the combined expression when compared to an appropriatecontrol plant. For example, an increase in the level of RNAi in theplant, plant part or the plant cell can comprise at least about a 1%,about a 1%-5%, about a 5% -10%, about a 10%-20%, about a 20%-30%, abouta 30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about70%-80%, about a 80%-90%, about a 90%-100% or greater increase in thelevel of RNAi in the plant, plant part, plant cell, or phloem whencompared to an appropriate control. In other embodiments, the increasein the level of RNAi in the plant, plant part, plant cell, or phloem cancomprise at least about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold, about a 10 fold-20 fold, about a 20 fold -30 fold, about a 30fold -40 fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about60 fold -70 fold, about 70 fold-80 fold, about a 80 fold-90 fold, abouta 90 fold-100 fold or greater increase in the level of RNAi in theplant, plant part, plant cell or phloem when compared to an appropriatecontrol. Methods to assay for an increase in the level of RNAi arediscussed elsewhere herein.

Non-limiting examples of methods and compositions disclosed herein areas follows:

-   1. An isolated polynucleotide comprising a nucleotide sequence    selected from the group consisting of:

(a) the nucleotide sequence comprising any one of SEQ ID NOS: 279, 302,281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290,291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343,344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297,298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328 or acomplement thereof;

(b) the nucleotide sequence comprising at least 90% sequence identity toany one of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284,285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337,338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311,312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324,325, 326, 327 or 328 or a complement thereof, wherein saidpolynucleotide encodes a silencing element having insecticidal activityagainst a Pentatomidae plant pest;

(c) the nucleotide sequence comprising at least 19 consecutivenucleotides of any one of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282,303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14,18 or 263 or a complement thereof, wherein said polynucleotide encodes asilencing element having insecticidal activity against a Pentatomidaeplant pest; and,

(d) the nucleotide sequence that hybridizes under stringent conditionsto the full length complement of the nucleotide sequence of a), whereinsaid stringent conditions comprise hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1× SSC at 60° C. to 65° C.,wherein said polynucleotide encodes a silencing element havinginsecticidal activity against a Pentatomidae plant pest.

-   2. The isolated polynucleotide of embodiment 1, wherein said    Pentatomidae plant pest is a N. viridula plant pest.-   3. An expression cassette comprising a heterologous polynucleotide    of embodiment 1 or 2 operably linked to a seed-preferred promoter.-   4. The expression cassette of embodiment 3, wherein said    polynucleotide is expressed as a double stranded RNA.-   5. The expression cassette of embodiment 3, wherein said    polynucleotide comprise a silencing element which is expressed as a    hairpin RNA.-   6. The expression cassette of embodiment 5, wherein the silencing    element comprises, in the following order, a first segment, a second    segment, and a third segment, wherein

a) said first segment comprises at least about 19 nucleotides having atleast 90% sequence complementarity to a target sequence set forth in SEQID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287,288, 289, 290, 291, 292, 17, 30, 34, 14, 18 or 263;

b) said second segment comprises a loop of sufficient length to allowthe silencing element to be transcribed as a hairpin RNA; and,

c) said third segment comprises at least about 19 nucleotides having atleast 85% complementarity to the first segment.

-   7. The expression cassette of embodiment 6, wherein said target    sequence comprises the sequences set forth any one of SEQ ID NOS:    284, 285, 286, 287, 288, 289, 290, 291, 292, 337, 338, 339, 340,    341, 342, 343 or 344 or a sequence having at least 90% sequence    identity to SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292,    337, 338, 339, 340, 341, 342, 343 or 344.-   8. The expression cassette of embodiment 6, wherein said expression    cassette comprises any one of SEQ ID NOS: 293, 294, 295, 296, 297,    298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328.-   9. The expression cassette of embodiment 3, wherein said    polynucleotide is flanked by a first operably linked convergent    promoter at one terminus of the polynucleotide and a second operably    linked convergent promoter at the opposing terminus of the    polynucleotide, wherein the first and the second convergent    promoters are capable of driving expression of the polynucleotide.-   10. A host cell comprising a heterologous expression cassette of any    one of embodiments 3-9.-   11. A plant cell having stably incorporated into its genome a    heterologous polynucleotide comprising a silencing element operably    linked to a seed-preferred promoter, wherein said silencing element,    when ingested by a Pentatomidae plant pest, reduces the level of    expression of any one of the target sequences set forth in SEQ ID    NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286,    287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338,    339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311,    312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323,    324, 325, 326, 327 or 328 in said Pentatomidae plant pest and    thereby controls the Pentatomidae plant pest.-   12. The plant cell of embodiment 11, wherein said silencing element    comprises

a) a fragment of at least 19 consecutive nucleotides of SEQ ID NOS: 279,302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289,290, 291, 292, 17, 30, 34, 14, 18 or 263 or a complement thereof; or,

b) the nucleotide sequence comprising at least 90% sequence identity toany one of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284,285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337,338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311,312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324,325, 326, 327 or 328 or a complement thereof,

wherein said silencing element, when ingested by a Pentatomidae plantpest, reduces the level of a target sequence in said Pentatomidae plantpest and thereby controls the Pentatomidae plant pest.

-   13. The plant cell of embodiment 12, wherein the Pentatomidae plant    pest is a N. viridula plant pest.-   14. The plant cell of any one of embodiment 11, 12 or 13, wherein    said silencing element comprises the sequences set forth in any one    of SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 305,    306, 307, 308, 309, 310, 311, 312, 17, 30, 34, 337, 338, 339, 340,    341, 342, 343 or 344 or a complement thereof.-   15. The plant cell of embodiment 11-14, wherein said plant cell    comprises the expression cassette of embodiment 9.-   16. The plant cell of any one of embodiments 11-14, wherein said    silencing element expresses a double stranded RNA.-   17. The plant cell of any one of embodiments 11-15, wherein said    silencing element expresses a hairpin RNA.-   18. The plant cell of embodiment 17, wherein said polynucleotide    comprising the silencing element comprises, in the following order,    a first segment, a second segment, and a third segment, wherein

a) said first segment comprises at least about 19 nucleotides having atleast 90% sequence complementarity to a target sequence set forth in SEQID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287,288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340,341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294,295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or328;

b) said second segment comprises a loop of sufficient length to allowthe silencing element to be transcribed as a hairpin RNA; and,

c) said third segment comprises at least about 19 nucleotides having atleast 85% complementarity to the first segment.

-   19. The plant cell of any one of embodiments 11-18, wherein said    plant cell is from a monocot.-   20. The plant cell of embodiment 19, wherein said monocot is maize,    barley, millet, wheat or rice.-   21. The plant cell of any one of embodiments 11-18, wherein said    plant cell is from a dicot.-   22. The plant cell of embodiment 21, wherein said plant is soybean,    canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, or    cotton.-   23. A plant or plant part comprising a plant cell of any one of    embodiments 11-22.-   24. A transgenic seed from the plant of embodiment 23, wherein said    transgenic seed comprises said heterologous polynucleotide    comprising said silencing element.-   25. A method of controlling a Pentatomidae plant pest comprising    feeding to a Pentatomidae plant pest a composition comprising a    silencing element, wherein said silencing element, when ingested by    said Pentatomidae plant pest, reduces the level of expression of any    one of the target Pentatomidae plant pest sequences set forth in SEQ    ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286,    287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18 or 263 and thereby    controls the Pentatomidae plant pest.-   26. The method of embodiment 25, wherein said silencing element    comprises

a) a fragment of at least 19 consecutive nucleotides of SEQ ID NOS: 279,302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289,290, 291, 292, 17, 30, 34, 14, 18 or 263 or a complement thereof; or,

b) the nucleotide sequence comprising at least 90% sequence identity toany one of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284,285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337,338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311,312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324,325, 326, 327 or 328 or a complement thereof, wherein said silencingelement, when ingested by a Pentatomidae plant pest, reduces the levelof a target sequence in said Pentatomidae plant pest and therebycontrols the Pentatomidae plant pest.

-   27. The method of embodiment 26, wherein said Pentatomidae plant    pest comprises a N. viridula plant pest. 28. The method of any one    of embodiments 26 or 27, wherein said silencing element comprises    the sequence set forth in any one of SEQ ID NOS: 284, 285, 286, 287,    288, 289, 290, 291, 292, 305, 306, 307, 308, 309, 310, 311, 312, 17,    30, 34, 337, 338, 339, 340, 341, 342, 343 or 344 or a complement    thereof.-   29. The method of any one of embodiments 25-28, wherein said    composition comprises a plant or plant part having stably    incorporated into its genome a polynucleotide comprising said    silencing element, wherein said silencing element is operably linked    to a seed-preferred promoter.-   30. The method of any one of embodiments 25-29, wherein said    silencing element comprises

a) a polynucleotide comprising the sense or antisense sequence of thesequence set forth in SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290,291, 292, 17, 30, 34, 14, 18, 263, 337, 338, 339, 340, 341, 342, 343,344, 305, 306, 307, 308, 309, 310, 311 or 312 or a complement thereof;or,

b) a polynucleotide comprising the sense or antisense sequence of asequence having at least 95% sequence identity to the sequence set forthin SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34,14, 18, 263, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308,309, 310, 311 or 312 or a complement thereof;

-   31. The method of any one of embodiments 25-30, wherein said    silencing element expresses a double stranded RNA.-   32. The method of any one of embodiments 25-30, wherein said    silencing element comprises a hairpin RNA.-   33. The method of embodiment 32, wherein said polynucleotide    comprising the silencing element comprises, in the following order,    a first segment, a second segment, and a third segment, wherein

a) said first segment comprises at least about 20 nucleotides having atleast 90% sequence complementarity to the target polynucleotide;

b) said second segment comprises a loop of sufficient length to allowthe silencing element to be transcribed as a hairpin RNA; and,

c) said third segment comprises at least about 20 nucleotides having atleast 85% complementarity to the first segment.

-   34. The method of any one of embodiments 29-30, wherein said    silencing element is flanked by a first operably linked convergent    promoter at one terminus of the silencing element and a second    operably linked convergent promoter at the opposing terminus of the    polynucleotide, wherein the first and the second convergent    promoters are capable of driving expression of the silencing    element.-   35. The method of embodiment 29, wherein said plant is a monocot.-   36. The method of embodiment 35, wherein said monocot is maize,    barley, millet, wheat or rice.-   37. The method of embodiment 29, wherein said plant is a dicot.-   38. The method of embodiment 37, wherein said plant is soybean,    canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, or    cotton.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 In Vitro Transcription dsRNA Screening Method

A high throughput survey of candidate genes from the stinkbug Nezaraviridula was performed for their potential utility as a target for RNAileading to mortality (insecticidal activity of RNAi). A library of over1000 expressed sequence tags was subjected to in vitro transcription andindividual samples tested against 2nd instar nymphs of N. viridula. Theinsects were fed the sample in an insect assay format. After 6 days, thenumber of dead nymphs was recorded. Table 1 provides the blast homology(Gene ID) of the various silencing elements (clone name) disclosedherein and also provides bioassay data demonstrating the insecticidalactivity of the various sequences when fed to N. viridula.

TABLE 1 6 day score clone name Gene ID #dead/10 inv1c.pk008.f8.f no hits10 inv1c.pk003.n13.f conserved hypothetical protein 10 inv1c.pk003.o24.fconserved hypothetical protein 7 inv1c.pk004.a3.f cathepsin L1 precursor9 inv1c.pk004.a23.f no hits 9 inv1c.pk004.b4.f forked protein 8inv1c.pk004.b6.f ribosomal protein L24e 9 inv1c.pk004.b17.f no hits 8inv1c.pk004.b23.f nonspecific lipid transfer protein/sterol 9 carrierprotein inv1c.pk004.c11.f soldier specific protein 7 inv1c.pk004.c12.fno hits 10 inv1c.pk004.d4.f no hits 8 inv1c.pk004.d16.f oligomycinsensitivity conferral 10 protein//ATP synthase inv1c.pk004.d17.f no hits10 inv1c.pk004.d19.f no hits 7 inv1c.pk004.d20.f no hits 9inv1c.pk004.e6.f mitochondrial protein PTCD3 10 inv1c.pk004.e11.fadapter molecule Crk 10 inv1c.pk004.e24.f cytochrome P450 10inv1c.pk004.f2.f no hits 8 inv1c.pk004.f10.f no hits 7 inv1c.pk004.f12.fno hits 8 inv1c.pk004.f17.f similar to dipteran sequences 7inv1c.pk004.f24.f no hits 8 inv1c.pk004.g13.f no hits 8inv1c.pk004.g20.f vertebrate homology 9 inv1c.pk004.g22.f no hits 10inv1c.pk004.g23.f no hits 8 inv1c.pk004.h18.f salivary protein 10inv1c.pk004.h20.f lin-52 homolog 8 inv1c.pk004.h21.f cyclin t 10inv1c.pk004.h23.f similar to complement component 1 q 9 subcomponentbinding protein-like protein inv1c.pk004.h24.f similar to prefoldinsubunit 10 inv1c.pk004.i1.f hsp70 10 inv1c.pk004.i4.f serine/threoninekinase 9 inv1c.pk004.i7.f no hits 9 inv1c.pk004.i14.f cytochrome P450 10inv1c.pk005.f6.f U6 snRNA-associated Sm-like protein 7 inv1c.pk005.f8.fNADH dehydrogenase subunit 2 10 inv1c.pk005.f20.f apolipprotein D 7inv1c.pk005.h1.f similar to Gag protein 10 inv1c.pk005.i21.f no hits 8inv1c.pk005.j11.f no hits 8 inv1c.pk005.j17.f Homo sapiens 3 BACRP11-666A9 9 inv1c.pk005.k12.f no hits 7 inv1c.pk005.l13.f no hits 10inv1c.pk005.m5.f no hits 10 inv1c.pk005.m16.f similar to translationinitiation factor 3, 8 subunit S8 inv1c.pk006.j24.f no hits 9inv1c.pk006.k4.f no hits 7 inv1c.pk006.k18.f acyl-CoA binding protein 7inv1c.pk006.k20.f E3 ubiquitin ligase/zinc finger protein 9inv1c.pk006.k21.f no hits 8 inv1c.pk006.l7.f nervana 3/similar to 7sodium/potassium-dependent atpase beta-2 subunit inv1c.pk006.m2.f nohits 10 inv1c.pk006.m13.f no hits 10 inv1c.pk006.o14.f no hits 10inv1c.pk006.p4.f ubiquinol-cytochrome c reductase 10 complex 11 kDaprotein inv1c.pk006.p8.f similar to ATPase inhibitor-like protein 10inv1c.pk006.p11.f 40S ribosomal protein S7 9 inv1c.pk006.p14.f similarto Drosophila and pea aphid 10 sequences inv1c.pk007.a5.f homology toinsect sequences 8 (Nasonia, Tribolium, Drosophila inv1c.pk007.b6.f nohits 9 inv1c.pk007.c6.f no hits 10 inv1c.pk007.c9.f conservedhypothetical protein 10 inv1c.pk007.d17.f putative ferritin 10inv1c.pk007.e5.f fatty acyl-CoA elongase 10 inv1c.pk007.e21.f aldehydedehydrogenase 9 inv1c.pk007.f1.f no hits 10 inv1c.pk007.f9.fbeta-tubulin 10 inv1c.pk007.f12.f no hits 10 inv1c.pk007.f19.fmitochondrial import receptor subunit 10 tom40 [Aedes aegypti]inv1c.pk007.f24.f no hits 9 inv1c.pk007.g6.f no hits 10inv1c.pk007.g17.f putative odorant-binding protein 10 precursorinv1c.pk007.h7.f no hits 7 inv1c.pk007.h11.f no hits 8 inv1c.pk007.h19.fno hits 7 inv1c.pk007.i7.f transposase 10 inv1c.pk007.i16.f venomprophenoloxidase-activating 10 protease inv1c.pk007.j14.f no hits 10inv1c.pk007.j19.f no hits 10 inv1c.pk007.j21.f conserved hypotheticalprotein 9 inv1c.pk007.j23.f succinate dehydrogenase, 7 cytochrome Bsmall subunit inv1c.pk007.j24.f no hits 8 inv1c.pk007.k17.f conservedhypothetical protein 9 inv1c.pk007.l5.f no hits 9 inv1c.pk007.l8.ftransmembrane protein, putative 9 inv1c.pk007.l11.f no hits 10inv1c.pk007.m6.f conserved hypothetical protein 10 inv1c.pk007.m21.f nohits 9 inv1c.pk007.o14.f proteasome beta subunit 7 inv1c.pk007.p17.f nohits 7 inv1c.pk008.c8.f ribosomal protein L35Ae 7 inv1c.pk008.c15.fsimilar to prohibitin 8 inv1c.pk008.c17.f no hits 7 inv1c.pk008.d1.fconserved hypothetical protein 9 inv1c.pk008.d3.f conserved hypotheticalprotein 7 inv1c.pk008.e11.f no hits 10 inv1c.pk008.e15.f no hits 10inv1c.pk008.f3.f conserved hypothetical protein 9 inv1c.pk008.f5.f nohits 8 inv1c.pk008.f8.f no hits 10 inv1c.pk008.f23.f no hits 10inv1c.pk008.g7.f no hits 7 inv1c.pk008.g22.f no hits 7 inv1c.pk008.h23.fputative ribosomal protein S26 10 inv1c.pk008.h24.f similar tomevalonate kinase 10 inv1c.pk008.i10.f no hits 9 inv1c.pk008.i21.fputative accessory gland protein 9 inv1c.pk008.j20.f similar toeukaryotic translation 8 initiation factor 3 subunit 2 betainv1c.pk008.k24.f no hits 7 inv1c.pk008.l11.f similar to phosphatase andactin 9 regulator inv1c.pk008.p18.f no hits 9 inv1c.pk009.b14.f no hits8 inv1c.pk009.b21.f ribosomal protein S20 7 inv1c.pk009.e9.f no hits 9inv1c.pk009.e10.f similar to sarco(endo)plasmic 7 reticulum-type calciumATPase inv1c.pk009.e17.f similar to serine/threonine protein 9 kinasedeath domain protein, pelle- like inv1c.pk009.f12.f no hits 10inv1c.pk009.f17.f no hits 7 inv1c.pk009.f19.f no hits 9 inv1c.pk009.g2.fno hits 8 inv1c.pk009.g21.f no hits 8 inv1c.pk009.h21.f no hits 10inv1c.pk009.i13.f no hits 10 inv1c.pk009.i24.f no hits 7inv1c.pk009.k4.f no hits 8 inv1c.pk009.k8.f conserved hypotheticalprotein 10 inv1c.pk010.a13.f no hits 7 inv1c.pk010.a16.f arginyl-tRNAsynthetase 7 inv1c.pk010.b7.f similar to tar RNA binding protein 10inv1c.pk010.e5.f no hits 7 inv1c.pk010.n9.f no hits 7 inv1c.pk010.n24.fno hits 10 inv1c.pk010.p16.f cytochrome c oxidase subunit II 7inv1c.pk010.p20.f no hits 8 inv1c.pk011.a20.f no hits 7inv1c.pk011.b11.f no hits 7

Sequences displaying insecticidal activity are advanced to confirmationand further evaluation of activity against other stinkbug pests. Theassay is scored for activity 6 days post infestation. The possiblescores are dead, severely stunted (little or now growth but alive),stunted (growth to second instar but not equivalent to controls), or noactivity. Samples demonstrating mortality or severe stunting areadvanced to confirmation.

Following confirmation, a simple dose response assay is performed withN. viridula. Samples for dose response assays is produced in the samemanner with the following modification; samples is further purifiedusing column purification prior to enzymatic treatment. Samples is alsonormalized to 0.5ug/ul and all samples are evaluated by gelelectrophoresis. Dose response assays is performed with the followingrates; 50, 25, 12, 6, 3, and 1.5 ppm

Example 2 Sequences Having Insecticidal Activity

DNA sequences which encode double stranded RNAs which were shown to haveinsecticidal activity against N. viridula using the assay described inExample 1 are set forth in SEQ ID NOS: 1-139.

Example 3 Transformation of Maize

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the silencing element of the invention operablylinked to either a tissue specific, tissue selective, or constitutivepromoter and the selectable marker gene PAT (Wohlleben et al. (1988)Gene 70:25-37), which confers resistance to the herbicide Bialaphos. Inone embodiment, the promoter employed is a seed-preferred promoter. Inone embodiment, the constructs will express a long double stranded RNAor a miRNA of the target sequence set forth in SEQ ID NOS: 1-292 or302-304 or a fragment thereof. In specific embodiments, the targetsequence comprises the sequences set forth in SEQ ID NOS: 278, 279, 280,281, 282, 283, 302, 303 or 304. Such a construct can be linked to apromoter active in maize. Alternatively, the selectable marker gene isprovided on a separate plasmid. Transformation is performed as follows.Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5cm target zone in preparation forbombardment.

A plasmid vector comprising the silencing element of interest operablylinked to either the tissue specific, tissue selective, or constitutivepromoter is made. This plasmid DNA plus plasmid DNA containing a PATselectable marker is precipitated onto 1.1 p.m (average diameter)tungsten pellets using a CaCl₂ precipitation procedure as follows: 100μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTAbuffer (1 μg total DNA); 100 μl 2.5 M CaCl₂; and,10 μl 0.1 M spermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates are bombarded at level #4 in a particle gun. Allsamples receive a single shot at 650 PSI, with a total of ten aliquotstaken from each tube of prepared particles/DNA.

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.

Plants are monitored and scored for the appropriate marker, such as thecontrol of a Pentatomidae plant pest, such as a N. viridula plant pest.For example, R₀ maize plants are fed to N. viridula 2nd instar nymphs.Contamination and larval quality are monitored. Larval mass andsurvivorship are recorded for analysis. A one-way ANOVA analysis and aDunnett's test is performed on the larval mass data to look forstatistical significance compared to an untransformed negative controlmaize plant diet. N. viridula 2n^(d) instar nymph stunting is measuredafter feeding on two events and compared to growth of larvae fed onnegative control plants.

In other assays, transgenic corn plants (R₀) generated are planted into10-inch pots containing Metromix soil after reaching an appropriatesize. After allowing the N. viridula 2^(nd) instar nymphs to feed on theplant, plants are removed from the soil and washed so that the relevantplant parts can be evaluated for larval feeding. Plant damage is ratedusing routine methods to score the level of damage.

Bombardment medium (560Y) comprises 4.0 g/1 N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/1thiamine HCl, 120.0 g/l sucrose, 1.0 mg/12,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000XSIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added aftersterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCl, 0.10 g/l pyridoxine HCl, and 0.40 g/lglycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositol,and 40.0 g/l sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 4 Agrobacterium-Mediated Transformation of Maize

For Agrobacterium-mediated transformation of maize with a silencingelement of the invention, the method of Zhao is employed (U.S. Pat. No.5,981,840, and PCT patent publication WO98/32326; the contents of whichare hereby incorporated by reference). Such a construct can, forexample, express a long double stranded RNA or a miRNA of the targetsequence set forth in SEQ ID NOS: 1-292 or 302-304. In one embodiment,the promoter employed is a seed-preferred promoter. In specificembodiments, the target sequence comprises the sequence set forth in SEQID NOS: 278, 279, 280, 281, 282, 283, 302, 303 or 304. Such a constructcan be linked to the dMMB promoter. Briefly, immature embryos areisolated from maize and the embryos contacted with a suspension ofAgrobacterium, where the bacteria are capable of transferring thepolynucleotide comprising the silencing element to at least one cell ofat least one of the immature embryos (step 1: the infection step). Inthis step the immature embryos are immersed in an Agrobacteriumsuspension for the initiation of inoculation. The embryos areco-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos are cultured on solid mediumfollowing the infection step. Following this co-cultivation period anoptional “resting” step is contemplated. In this resting step, theembryos are incubated in the presence of at least one antibiotic knownto inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step). Theimmature embryos are cultured on solid medium with antibiotic, butwithout a selecting agent, for elimination of Agrobacterium and for aresting phase for the infected cells. Next, inoculated embryos arecultured on medium containing a selective agent and growing transformedcallus is recovered (step 4: the selection step). The immature embryosare cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium are cultured on solid medium to regenerate the plants.Assays for insecticidal activity can be performed as described above inExample 3.

Example 5 Soybean Embryo Transformation Culture Conditions

Soybean embryogenic suspension cultures (cv. Jack) are maintained in 35ml liquid medium SB196 (see recipes below) on rotary shaker, 150 rpm,26° C. with cool white fluorescent lights on 16:8 hr day/nightphotoperiod at light intensity of 60-85 μE/m2/s. Cultures aresubcultured every 7 days to two weeks by inoculating approximately 35 mgof tissue into 35 ml of fresh liquid SB196 (the preferred subcultureinterval is every 7 days).

Soybean embryogenic suspension cultures are transformed with a plasmidcontaining the silencing element of the invention operably linked toeither a tissue specific, tissue selective, or constitutive promoter bythe method of particle gun bombardment (Klein et al. (1987) Nature,327:70). In one embodiment, the promoter employed is a seed-preferredpromoter. In one embodiment, the constructs will express a long doublestranded RNA or a miRNA of the target sequence set forth in SEQ ID NOS:1-292 or 302-304 or a fragment thereof. In specific embodiments, thetarget sequence comprises the sequences set forth in SEQ ID NOS: 278,279, 280, 281, 282, 283, 302, 303 or 304.

Soybean Embryogenic Suspension Culture Initiation

Soybean cultures are initiated twice each month with 5-7 days betweeneach initiation.

Pods with immature seeds from available soybean plants 45-55 days afterplanting are picked, removed from their shells and placed into asterilized magenta box. The soybean seeds are sterilized by shaking themfor 15 minutes in a 5% Clorox solution with 1 drop of ivory soap (95 mlof autoclaved distilled water plus 5 ml Clorox and 1 drop of soap). Mixwell. Seeds are rinsed using 2 1-liter bottles of sterile distilledwater and those less than 4 mm are placed on individual microscopeslides. The small end of the seed are cut and the cotyledons pressed outof the seed coat. Cotyledons are transferred to plates containing SB1medium (25-30 cotyledons per plate). Plates are wrapped with fiber tapeand stored for 8 weeks. After this time secondary embryos are cut andplaced into SB196 liquid media for 7 days.

Preparation of DNA for Bombardment

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene are used for bombardment.Plasmid DNA for bombardment are routinely prepared and purified usingthe method described in the Promega™ Protocols and Applications Guide,Second Edition (page 106). Fragments of the plasmids carrying thesilencing element of interest are obtained by gel isolation of doubledigested plasmids. In each case, 100 ug of plasmid DNA is digested in0.5 ml of the specific enzyme mix that is appropriate for the plasmid ofinterest. The resulting DNA fragments are separated by gelelectrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker MolecularApplications) and the DNA fragments containing silencing element ofinterest are cut from the agarose gel. DNA is purified from the agaroseusing the GELase digesting enzyme following the manufacturer's protocol.

A 50 μl aliquot of sterile distilled water containing 3 mg of goldparticles (3 mg gold) is added to 5 μg/μl DNA solution (either intactplasmid or DNA fragment prepared as described above), 50 μl 2.5M CaCl₂and 20 μl of 0.1 M spermidine. The mixture is shaken 3 min on level 3 ofa vortex shaker and spun for 10 sec in a bench microfuge. After a washwith 400 μl 100% ethanol the pellet is suspended by sonication in 40 μlof 100% ethanol. Five μl of DNA suspension is dispensed to each flyingdisk of the Biolistic PDS1000/HE instrument disk. Each 5 μl aliquotcontains approximately 0.375 mg gold per bombardment (i.e. per disk).

Tissue Preparation and Bombardment with DNA

Approximately 150-200 mg of 7 day old embryonic suspension cultures areplaced in an empty, sterile 60×15 mm petri dish and the dish coveredwith plastic mesh. Tissue is bombarded 1 or 2 shots per plate withmembrane rupture pressure set at 1100 PSI and the chamber evacuated to avacuum of 27-28 inches of mercury. Tissue is placed approximately 3.5inches from the retaining / stopping screen.

Selection of Transformed Embryos

Transformed embryos were selected either using hygromycin (when thehygromycin phosphotransferase, HPT, gene was used as the selectablemarker) or chlorsulfuron (when the acetolactate synthase, ALS, gene wasused as the selectable marker).

Hygromycin (HPT) Selection

Following bombardment, the tissue is placed into fresh SB196 media andcultured as described above. Six days post-bombardment, the SB196 isexchanged with fresh SB196 containing a selection agent of 30 mg/Lhygromycin. The selection media is refreshed weekly. Four to six weekspost selection, green, transformed tissue may be observed growing fromuntransformed, necrotic embryogenic clusters. Isolated, green tissue isremoved and inoculated into multiwell plates to generate new, clonallypropagated, transformed embryogenic suspension cultures.

Chlorsulfuron (ALS) Selection

Following bombardment, the tissue is divided between 2 flasks with freshSB196 media and cultured as described above. Six to seven dayspost-bombardment, the SB196 is exchanged with fresh SB196 containingselection agent of 100 ng/ml Chlorsulfuron. The selection media isrefreshed weekly. Four to six weeks post selection, green, transformedtissue may be observed growing from untransformed, necrotic embryogenicclusters. Isolated, green tissue is removed and inoculated intomultiwell plates containing SB196 to generate new, clonally propagated,transformed embryogenic suspension cultures.

Regeneration of Soybean Somatic Embryos into Plants

In order to obtain whole plants from embryogenic suspension cultures,the tissue must be regenerated.

Embryo Maturation

Embryos are cultured for 4-6 weeks at 26° C. in SB196 under cool whitefluorescent (Phillips cool white Econowatt F40/CW/RS/EW) and Agro(Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr photoperiod with lightintensity of 90-120 uE/m2s. After this time embryo clusters are removedto a solid agar media, SB166, for 1-2 weeks. Clusters are thensubcultured to medium SB103 for 3 weeks. During this period, individualembryos can be removed from the clusters and screened for theappropriate marker or the ability of the plant, when injected with thesilencing elements, to control the Pentatomidae plant pest or the N.viridula plant pest.

Embryo Desiccation and Germination

Matured individual embryos are desiccated by placing them into an empty,small petri dish (35×10 mm) for approximately 4-7 days. The plates aresealed with fiber tape (creating a small humidity chamber). Desiccatedembryos are planted into SB71-4 medium where they were left to germinateunder the same culture conditions described above. Germinated plantletsare removed from germination medium and rinsed thoroughly with water andthen planted in Redi-Earth in 24-cell pack tray, covered with clearplastic dome. After 2 weeks the dome is removed and plants hardened offfor a further week. If plantlets looked hardy they are transplanted to10″ pot of Redi-Earth with up to 3 plantlets per pot.

Media Recipes

SB 196 - FN Lite liquid proliferation medium (per liter) - MS FeEDTA -100x Stock 1 10 ml MS Sulfate - 100x Stock 2 10 ml FN Lite Halides -100x Stock 3 10 ml FN Lite P, B, Mo - 100x Stock 4 10 ml B5 vitamins (1ml/L) 1.0 ml 2,4-D (10 mg/L final concentration) 1.0 ml KNO3 2.83 gm(NH4)2SO4 0.463 gm Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8

FN Lite Stock Solutions

Stock # 1000 ml 500 ml 1 MS Fe EDTA 100x Stock Na₂ EDTA* 3.724 g  1.862g  FeSO₄—7H₂O 2.784 g  1.392 g  2 MS Sulfate 100x stock MgSO₄—7H₂O 37.0g 18.5 g MnSO₄—H₂O 1.69 g 0.845 g  ZnSO₄—7H₂O 0.86 g 0.43 g CuSO₄—5H₂O0.0025 g  0.00125 g   3 FN Lite Halides 100x Stock CaCl₂—2H₂O 30.0 g15.0 g KI 0.083 g  0.0715 g  CoCl₂—6H₂O 0.0025 g  0.00125 g   4 FN LiteP, B, Mo 100x Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 gNa₂MoO₄—2H₂O 0.025 g  0.0125 g  *Add first, dissolve in dark bottlewhile stirring

SB1 solid medium (per liter) comprises: 1 pkg. MS salts (Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000× stock; 31.5 g sucrose; 2 ml 2,4-D(20mg/L final concentration); pH 5.7; and, 8 g TC agar.

SB 166 solid medium (per liter) comprises: 1 pkg. MS salts(Gibco/BRL-Cat# 11117-066); 1 ml B5 vitamins 1000× stock; 60 g maltose;750 mg MgC12 hexahydrate; 5 g activated charcoal; pH 5.7; and, 2 ggelrite.

SB 103 solid medium (per liter) comprises: 1 pkg. MS salts(Gibco/BRL-Cat# 11117-066); 1 ml B5 vitamins 1000× stock; 60 g maltose;750 mg MgC12 hexahydrate; pH 5.7; and, 2 g gelrite.

SB 71-4 solid medium (per liter) comprises: 1 bottle Gamborg's B5 saltsw/sucrose (Gibco/BRL-Cat# 21153-036); pH 5.7; and, 5 g TC agar. 2,4-Dstock is obtained premade from Phytotech cat# D 295 -concentration is 1mg/ml.

B5 Vitamins Stock (per 100 ml) which is stored in aliquots at −20Ccomprises: 10 g myo-inositol; 100 mg nicotinic acid; 100 mg pyridoxineHCl; and, 1 g thiamine. If the solution does not dissolve quicklyenough, apply a low level of heat via the hot stir plate. ChlorsulfuronStock comprises 1mg/ml in 0.01 N Ammonium Hydroxide

Example 6 Expression of Silencing Elements Comprising siRNAs

SiRNAs were generated to target the cDNA sequence set forth in SEQ IDNOS: 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176,179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218,221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260,263, 266, 269, 273, and 276. Table 2 provides the clone name of thesilencing element and the closest homology for the target sequence (genename). Table 3 provides the clone name, the target cDNA, the sense andantisense siRNA sequence, and the respective SEQ ID NOS. Table 4provides the bioassays for each of the siRNAs shown in Table 3.

TABLE 2 Query Sequence Title (ID) gene name inv1c.pk003.j16.f conservedprotein of unknown function inv1c.pk003.j16.f conserved protein ofunknown function inv1c.pk004.b7.f cathepsin L inv1c.pk004.b7.f cathepsinL inv1c.pk004.b7.f cathepsin L inv1c.pk004.b7.f cathepsin Linv1c.pk004.c4.f mitochondrial porin inv1c.pk004.c4.f mitochondrialporin inv1c.pk004.c4.f mitochondrial porin inv1c.pk004.c4.fmitochondrial porin inv1c.pk004.c4.f mitochondrial porininv1c.pk004.c4.f mitochondrial porin inv1c.pk004.c4.f mitochondrialporin inv1c.pk004.c4.f mitochondrial porin inv1c.pk004.c4.fmitochondrial porin inv1c.pk004.c4.f mitochondrial porininv1c.pk004.f4.f reverse transcriptase inv1c.pk004.f4.f reversetranscriptase inv1c.pk004.f4.f reverse transcriptase inv1c.pk004.j14.fsugar transporter inv1c.pk004.k9.f glutathione s transferaseinv1c.pk004.k9.f glutathione s transferase inv1c.pk004.k9.f glutathiones transferase inv1c.pk005.a24.f cathepsin L-like proteaseinv1c.pk005.a24.f cathepsin L-like protease inv1c.pk005.b16.f synapsininv1c.pk005.b16.f synapsin inv1c.pk005.b16.f synapsin inv1c.pk005.b16.fsynapsin inv1c.pk005.b16.f synapsin inv1c.pk005.f20.f Apolipoprotein Dprecursor inv1c.pk005.f20.f Apolipoprotein D precursor inv1c.pk005.f20.fApolipoprotein D precursor inv1c.pk005.f20.f Apolipoprotein D precursorinv1c.pk005.h1.f nucleic acid binding protein inv1c.pk005.h1.f nucleicacid binding protein inv1c.pk005.h1.f nucleic acid binding proteininv1c.pk005.h1.f nucleic acid binding protein inv1c.pk005.h1.f nucleicacid binding protein inv1c.pk005.h1.f nucleic acid binding proteininv1c.pk005.h1.f nucleic acid binding protein inv1c.pk005.h23.f chitinsynthase 1 inv1c.pk005.j19.f conserved hypothetical proteininv1c.pk005.j19.f conserved hypothetical protein inv1c.pk005.k24.fcathepsin B inv1c.pk005.k24.f cathepsin B

TABLE 3(Note: the sense RNA primer sequence and the antisense RNA primersequences shown in table 3 were generated having 2 thymineresidues at the 3′ end.) SEQ ID NOS Approx. Target cDNA/ Query TargetTarget Sense Antisense sense siRNA Sequence No. Target Location cDNAStrand Strand siRNA/antisense number Title (ID) Bases Location (thirds)Sequence %CG siRNA siRNA siRNA  1 inv1c.pk003. 656 227 2 AATCAAGGTGTGGA34.8 UCAAGGUGUG UUUUCAGUCCA 140/141/142 j16.f CTGAAAATT GACUGAAAACACCUUGA  2 inv1c.pk003. 656 490 3 AATTGGTTGCTACAT 30.4 UUGGUUGCUAGAGAAUAUGUA 143/144/145 j16.f ATTCTCTT CAUAUUCUC GCAACCAA  3inv1c.pk004. 603 150 1 AAGAACGTCTTAGG 34.8 GAACGUCUUA UAUGCAUCCUA146/147/148 b7.f ATGCATATT GGAUGCAUA AGACGUUC  4 inv1c.pk004. 603 317 2AAGCAAGCACCTAC 43.5 GCAAGCACCU UGUGAAGGUAG 149/150/151 b7.f CTTCACATTACCUUCACA GUGCUUGC  5 inv1c.pk004. 603 412 3 AAACCAAGGTAGCT 43.5ACCAAGGUAG GAUCCACAGCU 152/153/154 b7.f GTGGATCTT CUGUGGAUC ACCUUGGU  6inv1c.pk004. 603 545 3 AATAATGGATGTGG 43.5 UAAUGGAUGU UCCGCCACCACA155/156/157 b7.f TGGCGGATT GGUGGCGGA UCCAUUA  7 inv1c.pk004. 688 133 1AAAAGGATACCACT 34.8 AAGGAUACCA AGUCCAAAGUG 158/159/160 c4.f TTGGACTTTCUUUGGACU GUAUCCUU  8 inv1c.pk004. 688 134 1 AAAGGATACCACTT 34.8AGGAUACCAC AAGUCCAAAGU 161/162/163 c4.f TGGACTTTT UUUGGACUU GGUAUCCU  9inv1c.pk004. 688 171 1 AAACCAAGACCCAG 47.8 ACCAAGACCC CUCCAGUCUGG164/165/166 c4.f ACTGGAGTT AGACUGGAG GUCUUGGU 10 inv1c.pk004. 688 176 1AAGACCCAGACTGG 43.5 GACCCAGACU UUCAACUCCAG 167/168/169 c4.f AGTTGAATTGGAGUUGAA UCUGGGUC 11 inv1c.pk004. 688 218 1 AACCAAGAAACTGG 39.1CCAAGAAACU CACUUUCCCAG 170/171/172 c4.f GAAAGTGTT GGGAAAGUG UUUCUUGG 12inv1c.pk004. 688 226 1 AACTGGGAAAGTGT 39.1 CUGGGAAAGU UUUCCGAACAC173/174/175 c4.f TCGGAAATT GUUCGGAAA UUUCCCAG 13 inv1c.pk004. 688 322 2AACTGAAATTGCCCT 3.91 CUGAAAUUGC UCAGUGAGGGC 176/177/178 c4.f CACTGATTCCUCACUGA AAUUUCAG 14 inv1c.pk004. 688 359 2 AAGCTTTCTTGTGAT 34.8GCUUUCUUGU UGAGGUAUCAC 179/180/181 c4.f ACCTCATT GAUACCUCA AAGAAAGC 15inv1c.pk004. 688 431 2 AATGATACGTGTGCT 34.8 UGAUACGUGU GUUCAAAGCAC182/183/184 c4.f TTGAACTT GCUUUGAAC ACGUAUCA 16 inv1c.pk004. 688 619 3AAGCATTAATGATG 34.8 GCAUUAAUGA ACACGUCCAUC 185/186/187 c4.f GACGTGTTTUGGACGUGU AUUAAUGC 17 inv1c.pk004. 696 368 2 AAAACTTTCTCAAA 30.4AACUUUCUCA CUGGUUCUUUG 188/189/190 f4.f GAACCAGTT AAGAACCAG AGAAAGUU 18inv1c.pk004. 696 379 2 AAAGAACCAGTTCC 34.8 AGAACCAGUU UGCAUUUGGAA191/192/193 f4.f AAATGCATT CCAAAUGCA CUGGUUCU 19 inv1c.pk004. 696 394 2AATGCATTCCCTTCA 34.8 UGCAUUCCCU UGAGAUUGAAG 194/195/196 f4.f ATCTCATTUCAAUCUCA GGAAUGCA 20 inv1c.pk004. 687 533 3 AACCTCTCCTCGTCT 52.2CCUCUCCUCG GACUCCAGACG 197/198/199 j14.f GGAGTCTT UCUGGAGUC AGGAGAGG 21inv1c.pk004. 663 212 1 AAAGAATTCACCTG 39.1 AGAAUUCACC GUAGGACCAGG200/201/202 k9.f GTCCTACTT UGGUCCUAC UGAAUUCU 22 inv1c.pk004. 663 531 3AATTCTGGAAGAAA 34.8 UUCUGGAAGA UGGUCCAUUUC 203/204/205 k9.f TGGACCATTAAUGGACCA UUCCAGAA 23 inv1c.pk004. 663 641 3 AACGTCTAGAAATG 39.1CGUCUAGAAA CUCUCACCAUU 206/207/208 k9.f GTGAGAGTT UGGUGAGAG UCUAGACG 24inv1c.pk005. 443 198 2 AATAAGAAACACGA 39.1 UAAGAAACAC GCCUGCUUCGU209/210/211 a24.f AGCAGGCTT GAAGCAGGC GUUUCUUA 25 inv1c.pk005. 443 271 2AAATGAAGAGCCAT 34.8 AUGAAGAGCC AGCCUAAAUGG 212/213/214 a24.f TTAGGCTTTAUUUAGGCU CUCUUCAU 26 inv1c.pk005. 680  17 1 AACTTCGAACCATCT 52.2CUUCGAACCA CCGGGGAGAUG 215/216/217 b16.f CCCCGGTT UCUCCCCGG GUUCGAAG 27inv1c.pk005. 680 119 1 AAGCTTCCTTCACTA 34.8 GCUUCCUUCA CAUUUGUAGUG218/219/220 b16.f CAAATGTT CUACAAAUG AAGGAAGC 28 inv1c.pk005. 680 156 1AAGGTTCAGCTCCG 52.2 GGUUCAGCUC AGAUCCCCGGA 221/222/223 b16.f GGGATCTTTCGGGGAUCU GCUGAACC 29 inv1c.pk005. 680 540 3 AATCGACGACCAAA 34.8UCGACGACCA UCAGUAUUUUG 224/225/226 b16.f ATACTGATT AAAUACUGA GUCGUCGA 30inv1c.pk005. 680 569 3 AATACTTCAGAGTA 39.1 UACUUCAGAG UACGCCGUACU227/228/229 b16.f CGGCGTATT UACGGCGUA CUGAAGUA 31 inv1c.pk005. 662  46 1AAAATGAGAGCTAC 39.1 AAUGAGAGCU GCAGUACGUAG 230/231/232 f20.f GTACTGCTTACGUACUGC CUCUCAUU 32 inv1c.pk005. 662 316 2 AAATACCATTACAC 30.4AUACCAUUAC AUGUCCUGUGU 233/234/235 f20.f AGGACATTT ACAGGACAU AAUGGUAU 33inv1c.pk005. 662 387 2 AAGTGTTGCTGGAA 39.1 GUGUUGCUGG CUUGAUGUUCC236/237/238 f20.f CATCAAGTT AACAUCAAG AGCAACAC 34 inv1c.pk005. 662 605 3AATGCCCAGCAGAA 47.8 UGCCCAGCAG GGUUGGUUUCU 239/240/241 f20.f ACCAACCTTAAACCAACC GCUGGGCA 35 inv1c.pk005. 628 143 1 AAATACCACAGCCA 34.8AUACCACAGC UUAUUGCUGGC 242/243/244 h1.f GCAATAATT CAGCAAUAA UGUGGUAU 36inv1c.pk005. 628 144 1 AATACCACAGCCAG 34.8 UACCACAGCC AUUAUUGCUGG245/246/247 h1.f CAATAATTT AGCAAUAAU CUGUGGUA 37 inv1c.pk005. 628 192 1AAGCCTCCGGTACCT 52.2 GCCUCCGGUA ACCUUGAGGUA 248/249/250 h1.f CAAGGTTTCCUCAAGGU CCGGAGGC 38 inv1c.pk005. 628 288 2 AATCTTATCGGACA 34.8UCUUAUCGGA ACUGGUUUGUC 251/252/253 h1.f AACCAGTTT CAAACCAGU CGAUAAGA 39inv1c.pk005. 628 556 3 AAAAATATCCATTG 30.4 AAAUAUCCAU ACAGUGGCAAU254/255/256 h1.f CCACTGTTT UGCCACUGU GGAUAUUU 40 inv1c.pk005. 628 557 3AAAATATCCATTGCC 30.4 AAUAUCCAUU AACAGUGGCAA 257/258/259 h1.f ACTGTTTTGCCACUGUU UGGAUAUU 41 inv1c.pk005. 628 558 3 AAATATCCATTGCCA 30.4AUAUCCAUUG AAACAGUGGCA 260/261/262 h1.f CTGTTTTT CCACUGUUU AUGGAUAU 42inv1c.pk005. 647 301 2 AAGGATGGGATGTG 47.8 GGAUGGGAUG CUCGGAACACA263/264/265 h23.f TTCCGAGTT UGUUCCGAG UCCCAUCC 43 inv1c.pk005. 597 172 1AAGATGGGGGGATG 47.8 GAUGGGGGGA CGUACAUCAUC 266/267/268 j19.f ATGTACGTTUGAUGUACG CCCCCAUC 44 inv1c.pk005. 597 377 2 AAGAACATCCACAG 43.5GAACAUCCAC GGUUCUCCUGU 269/270/271 j19.f GAGAACCTT AGGAGAACC GGAUGUUC 45inv1c.pk005. 593 27 1 AAGACTCTATTAATA 30.4 GACUCUAUUA GCUGGAUAUUA272/273/274 k24.f TCCAGCTT AUAUCCAGC AUAGAGUC 46 inv1c.pk005. 593 132 1AAATGGAAAGCTGG 43.5 AUGGAAAGCU GUUCUGCCCAG 275/276/277 k24.f GCAGAACTTGGGCAGAAC CUUUCCAU

TABLE 4 Bioassay-1 Bioassay-1 Bioassay-2 Bioassay-3 100 ppm 100 ppm 100ppm 100 ppm Bioassay-4 siRNA Query Sequence (4 day (5 day (5 day (5 day25 ppm Bioassay-5 Bioassay-6 number Title (ID) score) score) score)Comment score) 5 day 25 ppm 50 ppm 1 inv1c.pk003.j16.f 3/9  10/10  8/10ND 2 inv1c.pk003.j16.f 4/11 10/10  1/10 ND 3 inv1c.pk004.b7.f 11/12 10/10  ND ND 4 inv1c.pk004.b7.f 7/11 10/10  0/10 5 inv1c.pk004.b7.f 6/9 9/10 0/10 6 inv1c.pk004.b7.f 5/11 3/10 7 inv1c.pk004.c4.f 3/9  1/10 8inv1c.pk004.c4.f 3/10 0/10 9 inv1c.pk004.c4.f 4/10 4/10 (5 stunted) 0/1010 inv1c.pk004.c4.f 3/10 5/10 (3 stunted) 1/10 11 inv1c.pk004.c4.f 4/102/10 12 inv1c.pk004.c4.f 5/9  0/10 13 inv1c.pk004.c4.f 7/10 1/10 14inv1c.pk004.c4.f 4/10 2/10 15 inv1c.pk004.c4.f 5/9  1/10 16inv1c.pk004.c4.f 6/10 0/10 17 inv1c.pk004.f4.f 5/10 3/10 (2 stunted) 18inv1c.pk004.f4.f 2/11 3/10 (2 stunted) 19 inv1c.pk004.f4.f 6/9  0/10 20inv1c.pk004.j14.f 6/10 0/10 21 inv1c.pk004.k9.f 10/10 0/10 22inv1c.pk004.k9.f  6/11 1/10 23 inv1c.pk004.k9.f  3/10 2/10 24inv1c.pk005.a24.f 10/10 0/10 25 inv1c.pk005.a24.f 0/10 26inv1c.pk005.b16.f 0/10 Significant growth in survivors 27inv1c.pk005.b16.f 5/10 Significant growth in survivors 28inv1c.pk005.b16.f 5/10 Significant growth in survivors 29inv1c.pk005.b16.f 4/10 Significant growth in survivors 30inv1c.pk005.b16.f 0/10 31 inv1c.pk005.f20.f 9/10 No growth 1/10 32inv1c.pk005.f20.f 10/10  Some growth 1/10 before death 33inv1c.pk005.f20.f 10/10  Growth 2/10 (survivors 4/10 8/10 before deathstunted) 34 inv1c.pk005.f20.f 4/10 Growth before death 35inv1c.pk005.h1.f 7/10 Growth 1/10 (some before death stunting) 36inv1c.pk005.h1.f 10/10  some growth 1/10 before death 37inv1c.pk005.h1.f 10/10  Growth 0/10 before death 38 inv1c.pk005.h1.f10/10  no growth 1/10 39 inv1c.pk005.h1.f 1/10 40 inv1c.pk005.h1.f 8/10little growth 0/10 41 inv1c.pk005.h1.f 6/10 some growth 42inv1c.pk005.h23.f 7/10 No growth 0/10 43 inv1c.pk005.j19.f 10/10  Nogrowth 2/10 44 inv1c.pk005.j19.f 1/10 45 inv1c.pk005.k24.f 0/10 46inv1c.pk005.k24.f 0/10 5/10

Example 7 Constructs Expressing siRNAs

siRNAs designed to target the cDNA sequence set forth in SEQ ID NOS:140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179,182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221,224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263,266, 269, 272, and 275 can be engineered to be expressed in planta. Theconstruct can comprise, for example, the maize ubiquitinpromoter/5′UTR/1^(st) intron operably linked to a sequence comprisingSEQ ID NO: 141 which is operably linked to the ADH1 intron followed bythe sequence comprising SEQ ID NO: 142. It is recognized that any of thesiRNA described in Example 6 can be generated employing a simlarconstruct design.

Example 8 Generation of Silencing Constructs for in vivo TestingExperiments

The activity of 9 dsRNAs listed in Table 1, was confirmed on repeatedtesting and the target genes advanced for further evaluation in inplanta assays. For this purpose, 2 different types of constructs wereassembled. In one, 2 SGSB target gene fragments, separated by aspiceosomal intron, were assembled in opposite orientations with respectto each other to produce a hairpin RNA. In planta produced hairpin RNAsare expected to be processed to yield siRNAs which upon uptake intoinsects, mediate RNAi inhibition of SGSB target gene expression. In thesecond, small 21-mer SGSB gene sequences are incorporated into a microRNA backbone to produce an artificial pre-miRNA. Processing of thepre-miRNA in vivo releases the 21-nt miRNA that targets the SGSB genefor silencing. Hairpin constructs for in vivo expression and testing ofdsRNAs were assembled via Gateway technology using procedures andpractices well known to those skilled in the art of molecular biology.Target gene fragments were generated by PCR using gene specific senseand antisense primers containing Gateway attB4 (CAACTTTGTATAGAAAAGTTG(SEQ ID NO: 345)) and attB3 (CAACTTTGTATAATAAAGTTG (SEQ ID NO: 346))sequences, respectively. The amplified DNA fragments were recombinedinto the pDONR vector, PHP36164 containing attP4-attP3 sites in areaction catalyzed by BP Clonase. The resultant entry clones containingtarget gene fragments flanked by attL 4 and attL3 sites were then usedto generate an expression construct by performing 2 sequential LRrecombination reactions, first with the vector pKB499 and then with thevector PHP25224. The former destination vector contains the 193 bpintron2 fragment of the potato LS1 gene flanked by attR4-R3 sites at the5′ end and attR3-R4 sites at the 3′ end. LR recombination yields ahairpin segment comprised of sense and antisense target gene fragmentsseparated by an intron loop. In planta expression is regulated byplacement of the appropriate regulatory elements, promoter sequencesupstream and termination sequences downstream, of the hairpin segment.In this particular example, promoter sequences are provided by a 1946 bpsoybean ubiquitin promoter-5′ UTR-Intronl fragment and terminationsequences are provided by an 888bp 3′ fragment of the Arabidopsisubiquitin10 gene. Other promoter sequences providing constitutive orappropriate tissue specific expression may additionally be used. Thefinal plant expression construct is produced by a second LR reaction inwhich the entire hairpin cassette is moved into a vector (PHP25224)which provides a plant selectable marker (herbicide resistantacetolactate synthase gene) for stable transformation experiments. InTable 5, the 9 entries correspond to hairpin constructs that wereassembled and tested in soybean embryos for efficacy against SouthernGreen Stinkbug (SGSB).

TABLE 5 Hairpin constructs for SGSB target gene silencing Construct SEQID NO SEQ ID Fragment Fragment (without Gene ID SEQ length (bp) NOLocation SEQ ID NO Construct promoter) inv1c.pk004.e6.f:fis 1054 278 2-537 284 PHP49713 293 inv1c.pk004.h20.f:fis 861 279 72-677 285PHP48181 294 inv1c.pk004.h20.f:fis 861 279 72-834 286 pKB505 295inv1c.pk004.h20.f:fis 861 279 72-439 287 pKB506 296 inv1c.pk004.i1.f:fis992 280 27-511 288 PHP48183 297 inv1c.pk004.i1.f:fis 992 280 488-938 289 pKB508 298 inv1c.pk008.m9.f:fis 858 281  2-800 290 PHP49450 299inv1c.pk011.f6.f:fis 792 282 19-594 291 PHP49451 300inv1c.pk010.g13.f:fis 643 283  4-785 292 PHP49480 301

Silencing constructs encoding artificial microRNAs (amiRNAs) that wouldhave the ability to silence Southern Green Stinkbug genes were designedlargely according to rules described in Schwab R, et al. (2005) Dev Cell8: 517-27. To summarize, microRNA sequences are 21 nucleotides inlength, start at their 5′-end with a “U”, display 5′ instabilityrelative to their star sequence which is achieved by including a C or Gat position 19, and their 10th nucleotide is either an “A” or an “U”. Anadditional requirement for artificial microRNA design was that theamiRNA have a high free delta-G as calculated using the ZipFoldalgorithm (Markham, N. R. & Zuker, M. (2005) Nucleic Acids Res. 33:W577-W581.) Optionally, a one base pair change was added to the 5′portion of the amiRNA so that the sequence differed from the targetsequence by one nucleotide. The amiRNAs that were used to silence SGSBgenes are given in Table 6.

TABLE 6 amiRNA Sequences Target amiRNA amiRNA SEQ SEQ ID precursorGENE ID ID NO amiRNA Sequence NO Nv-MCS Frg1 inv1c.pk005.h23.f 302taagtaccatgtccaacgcca 305 Nv-MCS Frg2 inv1c.pk005.h23.f 302tattacaataactgaccaccc 306 Nv-MMitpro2 inv1c.pk004.e6.f:fis 278tcctactacatatttccaccc 307 Nv-MitprotCD3 inv1c.pk004.e6.f:fis 278tattccttctatcttctccca 308 Nv-Madapmol2 inv1c.pk004.e11.f:fis 303taaagtatattaataattctt 309 Nv-MadadapCRK1 inv1c.pk004.e11.f:fis 303ttactatcttcccttacacaa 310 Nv-MNH1A inv1c.pk004.d17.f:fis 304tacgaagagataacacaagat 311 Nv-MNH1B inv1c.pk004.d17.f:fis 304taacaaaacaaaaaaaaactg 312

“Star sequences” are those that base pair with the amiRNA sequences inthe precursor RNA, to form imperfect stem structures. To form a perfectstem structure the star sequence would be the exact reverse complementof the amiRNA. The soybean precursor sequence miR159 as described inZhang, B. at al. (2008) Planta 229:161-182 was folded with MFold (M.Zuker (2003) Nucleic Acids Res. 31: 3406-15; and D. H. Mathews, J. etal. (1999) J. Mol. Biol. 288: 911-940). The miRNA sequence was thenreplaced with the amiRNA sequence and the endogenous star sequence wasreplaced with the exact reverse complement of the amiRNA. Changes in theartificial star sequence were introduced so that the structure of thestem would remain the same as the endogenous structure. The alteredsequence was then folded with mfold and the original and alteredstructures were compared by eye. If necessary, further alternations tothe artificial star sequence were introduced to maintain the originalstructure. The DNA sequences corresponding to the artificial starsequences that were used to silence the desired target genes are shownin Table 7.

TABLE 7 amiRNA Star Sequences amiRNA SEQ ID precursor GENE IDamiRNA Sequence NO Nv-MCS Frg1-Star inv1c.pk005.h23.ftggcgttggactaggtacttt 313 Nv-MCS Frg2-Star inv1c.pk005.h23.fgggtggtcagtatttgtaatt 314 Nv-MMitpro2-Star inv1c.pk004.e6.f:fisgggtggaaataattagtaggt 315 Nv-MitprotCD3-Star inv1c.pk004.e6.f:fistgggagaagatcaaaggaatt 316 Nv-Madapmol2-Star inv1c.pk004.e11.f:fisaagaattattataatactttt 317 Nv-MadadapCRK1-Star inv1c.pk004.e11.f:fisttgtgtaagggttgatagtat 318 Nv-MNH1A-Star inv1c.pk004.d17.f:fisatcttgtgttaaatcttcgtt 319 Nv-MNH1B-Star inv1c.pk004.d17.f:fiscagttttttttgcttttgttt 320

The soybean genomic miRNA precursor gene, miR159, was converted toamiRNA precursors by DNA synthesis (Genscript; Piscataway, NJ). DNAfragments were synthesized with flanking Avrll and Hpal sites and werecloned by restriction enzyme digestion followed by DNA ligationdownstream of the GmUbiquitin promoter-5′UTR-Intronl fragment in theUBQ-Kozack OXOXalt7 vector. LR recombination reaction between thisintermediate and the vector QC479i produced the eight final plantexpression constructs given in Table 8.

TABLE 8 amiRNA Precursors and Expression Constructs amiRNA TargetConstruct amiRNA precursor Sequence SEQ ID precursor SEQ ID SEQ IDExpression NO (with GENE ID length NO Target Sequence NO Constructpromoter) inv1c.pk005.h23.f 976 bp 321 tggcgttggacatggtactta 337PHP44230 329 inv1c.pk005.h23.f 977 bp 322 gggtggtcagttattgtaata 338PHP44231 330 inv1c.pk004.e6.f:fis 966 bp 323 gggtggaaatatgtagtagga 339PHP44770 331 inv1c.pk004.e6.f:fis 966 bp 324 tgggagaagatagaaggaata 340PHP44771 332 inv1c.pk004.e11.f:fis 966 bp 325 aagaattattaatatacttta 341PHP44772 333 inv1c.pk004.e11.f:fis 966 bp 326 ttgtgtaagggaagatagtaa 342PHP44773 334 inv1c.pk004.d17.f:fis 966 bp 327 atcttgtgttatctcttcgta 343PHP44789 335 inv1c.pk004.d17.f:fis 966 bp 328 cagtttttttttgttttgtta 344PHP44790 336

The SEQ ID NOS for the various target genes advanced for furtherevaluation in in planta assays are summarized in Table 9.

TABLE 9 Fragments Target of Target Sequences Encoding Silencing SEQ IDSequences Silencing Constructs for Target Elements for Target SequenceClone NO SEQ ID NO Sequence SEQ ID NO SEQ ID NO inv1c.pk004.d17.f:fis304 14, 343, 344 335 (amiRNA precursor sequence with 311 (miRNA)promoter) 312 (miRNA) 336 (amiRNA precursor sequence with 327 (miRNAprecursor sequence) promoter) 328 (miRNA precursor sequence)inv1c.pk004.e6.f:fis 278 17, 284, 339, 293 (hairpin RNA constructwithout 284 (hairpin RNA) 340 promoter) 307 (miRNA) 331(amiRNA precursorsequence with 308 (miRNA) promoter) 323 (miRNA precursor sequence) 332(amiRNA precursor sequence with 324 (miRNA precursor sequence) promoter)inv1c.pk004.e11.f:fis 303 18, 341, 342 333 (amiRNA precursor sequencewith 309 (miRNA) promoter) 310 (miRNA) 334 (amiRNA precursor sequencewith 325 (miRNA precursor sequence) promoter) 326 (miRNA precursorsequence) inv1c.pk004.h20.f:fis 279 30, 285, 286, 294 (hairpin RNAconstruct without 285 (hairpin RNA) 287 promoter) 286 (hairpin RNA) 295(hairpin RNA construct without 287 (hairpin RNA) promoter) 296 (hairpinRNA construct without promoter) inv1c.pk004.i1.f:fis 280 34, 288, 289297 (hairpin RNA construct without 288 (hairpin RNA) promoter) 289(hairpin RNA) 298 (hairpin RNA construct without promoter)inv1c.pk005.h23.f 302 263, 337, 329 (amiRNA precursor sequence with 264(sense siRNA, RNA sequence) 338 promoter) 265 (anti-sense siRNA, RNAsequence) 330 (amiRNA precursor sequence with 305 (miRNA) promoter) 306(miRNA) 321 (miRNA precursor sequence) 322 (miRNA precursor sequence)inv1c.pk008.m9.f:fis 281 290 299 (hairpin RNA construct without 290(hairpin RNA) promoter) inv1c.pk010.g13.f:fis 283 292 301 (hairpin RNAconstruct without 292 (hairpin RNA) promoter) inv1c.pk011.f6.f:fis 282291 300 (hairpin RNA construct without 291 (hairpin RNA) promoter)

Example 9 Transformation of Somatic Soybean Embryo Cultures CultureConditions:

Soybean embryogenic suspension cultures (cv. Jack) were maintained in 35mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. withcool white fluorescent lights on 16:8 hr day/night photoperiod at lightintensity of 60-85 μE/m2/s. Cultures were sub-cultured every 7 days totwo weeks by inoculating approximately 35 mg of tissue into 35 mL offresh liquid SB196 (the preferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures were transformed with thesoybean expression plasmids described in Example 8 by the method ofparticle gun bombardment (Klein et al., Nature, 327:70 (1987)) using aDuPont Biolistic PDS1000/HE instrument (helium retrofit) for alltransformations.

Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures were initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plants45-55 days after planting were picked, removed from their shells andplaced into a sterilized magenta box. The soybean seeds were sterilizedby shaking them for 15 min in a 5% Clorox solution with 1 drop of ivorysoap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1drop of soap, mixed well). Seeds were rinsed using 2 1-liter bottles ofsterile distilled water and those less than 4 mm were placed onindividual microscope slides. The small end of the seed was cut and thecotyledons pressed out of the seed coat. Cotyledons were transferred toplates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks,then transferred to SB1 for 2-4 weeks. Plates were wrapped with fibertape. After this time secondary embryos were cut and placed into SB196liquid media for 7 days.

Preparation of DNA for Bombardment:

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene may be used for bombardment.In the present example, pDNAs were isolated from bacterial transformantsusing a Qiagen mini-prep kit. DNA concentrations were determined by UVabsorbance. Each silencing construct and hygromycin selectable markerplasmid (PHP18956) were combined in a 9:1 weight ratio to give a 1 ug/ulDNA solution.

A 50 μL aliquot of sterile distilled water containing 1 mg of goldparticles was added to 5 μL of a 1 μg/μL DNA solution (intact silencingand selectable marker plasmids as described above), 50 μL 2.5M CaCl₂ amd20 μL of 0.1 M spermidine. The mixture was pulsed 5 times on level 4 ofa vortex shaker and spun for 5 sec in a bench microfuge. After a washwith 150 μL of 100% ethanol, the pellet was suspended by sonication in85 μl of 100% ethanol. Five μL of DNA suspension was dispensed to eachflying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μLaliquot contained approximately 0.058 mg gold particles per bombardment(i.e., per disk).

Tissue Preparation and Bombardment with DNA:

Approximately 100-150 mg of 7 day old embryonic suspension cultures wereplaced in an empty, sterile 60×15 mm petri dish and the dish was placedinside of an empty 150×25mm Petri dish. Tissue was bombarded 1 shot perplate with membrane rupture pressure set at 650 PSI and the chamber wasevacuated to a vacuum of 27-28 inches of mercury. Tissue was placedapproximately 2.5 inches from the retaining /stopping screen.

Selection of Transformed Embryos:

Transformed embryos were selected using hygromycin as the selectablemarker.

Specifically, following bombardment, the tissue was placed into freshSB196 media and cultured as described above. Six to eight dayspost-bombardment, the SB196 is exchanged with fresh SB196 containing 30mg/L hygromycin. The selection media was refreshed bi-weekly. Four tosix weeks post-selection, green, transformed tissue was observed growingfrom untransformed, necrotic embryogenic clusters. Isolated, greentissue was removed and inoculated into multi-well plates to generatenew, clonally propagated, transformed embryogenic suspension cultures.

Embryo Maturation:

Transformed embryogenic clusters were cultured for 1-3 weeks at 26 ° C.in SB196 under cool white fluorescent (Phillips cool white EconowattF40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hrphotoperiod with light intensity of 90-120 μE/m²s. Embryo clusters werethen removed to SB228 (SHaM) liquid media, 35 ml in 250ml Erlenmeyerflask, for 2-3 weeks. Tissue cultured in SB228 was maintained on arotary shaker, 130 rpm, 26° C. with cool white fluorescent lights on16:8 hr day/night photoperiod at light intensity of 60-85 82 E/m2/s.After this time, embryos were harvested and used in stinkbug feedingassays.

Media Recipes:

SB 196 - FN Lite Liquid Proliferation Medium, pH 5.8 (per liter) MSFeEDTA - 100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN LiteHalides - 100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5vitamins (1 mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO₃2.83 gm (NH₄)₂SO₄ 0.463 gm asparagine 1.0 gm sucrose (1%) 10 gm

FN Lite Stock Solutions

Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na₂ EDTA* 3.724 g 1.862 g  FeSO₄—7H₂O 2.784 g  1.392 g  2 MS Sulfate 100x stock MgSO₄—7H₂O37.0 g 18.5 g MnSO₄—H₂O 1.69 g 0.845 g  ZnSO₄—7H₂O 0.86 g 0.43 gCuSO₄—5H₂O 0.0025 g  0.00125 g   3 FN Lite Halides 100x Stock CaCl₂—2H₂O30.0 g 15.0 g KI 0.083 g  0.0715 g  CoCl₂—6H₂O 0.0025 g  0.00125 g   4FN Lite P, B, Mo 100x Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 gNa₂MoO₄—2H₂O 0.025 g  0.0125 g  *Add first, dissolve in dark bottlewhile stirring

SB1 Solid Medium, pH5.7 (Per Liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

31.5 g glucose

2 mL 2,4-D (20 mg/L final concentration)

8 g TC agar

SB199 Solid Medium (per liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

30g Sucrose

4 ml 2,4-D (40 mg/L final concentration)

pH 7.0

2 gm Gelrite

SB 71-4 Solid Medium (per liter)

1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL—Cat. No. 21153-036)

pH 5.7

5 g TC agar

2,4-D Stock

Obtain premade from Phytotech Cat. No. D 295—concentration 1 mg/mL

B5 Vitamins Stock (per 100 mL)

Store aliquots at −20° C.

10 g myo-inositol

100 mg nicotinic acid

100 mg pyridoxine HCl

1 g thiamine

If the solution does not dissolve quickly enough, apply a low level ofheat via the hot stir plate.

SB 228 - Soybean Histodifferentiation & Maturation (SHaM) (per liter)DDI H2O 600 ml FN-Lite Macro Salts for SHaM 10X 100 ml MS Micro Salts1000x 1 ml MS FeEDTA 100x 10 ml CaCl 100x 6.82 ml B5 Vitamins 1000x 1 mlL-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mlpH 5.8 Autoclave Add to cooled media (≦30 C.): *Glutamine (Final conc.30 mM) 4% 110 ml *Note: Final volume will be 1010 ml after glutamineaddition..

FN-lite Macro for SHAM 10×-Stock #1 (per liter)

(NH₄)2SO₄ (Ammonium Sulfate) 4.63 g KNO₃ (Potassium Nitrate) 28.3 gMgSO₄*7H₂0 (Magnesium Sulfate Heptahydrate)  3.7 g KH₂PO₄ (PotassiumPhosphate, Monobasic) 1.85 g Bring to volume Autoclave

MS Micro 1000×- Stock #2 (per 1 liter)

H₃BO₃ (Boric Acid)  6.2 g MnSO₄*H₂O (Manganese Sulfate Monohydrate) 16.9 g ZnSO4*7H20 (Zinc Sulfate Heptahydrate)  8.6 g Na₂MoO₄*2H20(Sodium Molybdate Dihydrate)  0.25 g CuSO₄*5H₂0 (Copper SulfatePentahydrate) 0.025 g CoCl₂*6H₂0 (Cobalt Chloride Hexahydrate) 0.025 gKI (Potassium Iodide) 0.8300 g  Bring to volume Autoclave

FeEDTA 100×- Stock #3 (per liter)

Na₂EDTA* (Sodium EDTA) 3.73 g FeSO₄*7H₂0 (Iron Sulfate Heptahydrate)2.78 g *EDTA must be completely dissolved before adding iron Bring toVolume Solution is photosensitive. Bottle(s) should be wrapped in foilto omit light. Autoclave

Ca 100×- Stock #4 (per liter)

CaCl₂*2H₂0 (Calcium Chloride Dihydrate) 44 g Bring to Volume Autoclave

B5 Vitamin 1000×- Stock #5 (per liter)

Thiamine*HCl 10 g  Nicotinic Acid 1 g Pyridoxine*HCl 1 g Myo-Inositol100 g  Bring to Volume Store frozen

4% Glutamine-Stock #6 (per liter)

DDI water heated to 30° C. 900 ml L-Glutamine  40 g Gradually add whilestirring and applying low heat. Do not exceed 35° C. Bring to VolumeFilter Sterilize Store frozen* *Note: Warm thawed stock in 31° C. bathto fully dissolve crystals

It is recognized that the experiments set forth in example 9 can beemployed with silencing elements operably linked to a seed-preferredpromoter, such as, for example, those provided by theb-conglycinin-alpha (Genbank accession GU723691), Kunitz trypsininhibitor 3 (AF233296), or the glycinin-1 (AB353075.1) genes.

Example 10 Assay of Transgenic Soybean Embryos for Efficacy AgainstSouthern Green Stinkbug

Cultures of SHaM maturated embryos, as described in Example 9, wereharvested by filtration and used in feeding bioassays with 2^(nd) instarsouthern green stinkbugs. A typical soy embryo transformation experimentyielded 20 to 30 independent events that were each evaluated in 4replicate assays. Each assay was set up in a 35 mm petri dish thatcontained a moistened Whatman filter disc and a H2O soaked cotton pelletalong with 450-500 mg of soy embryo tissue. Embryo samples were infestedwith 5-2^(nd) instar SGSBs, and the petri plate was covered andincubated at 27C, 65% RH for 4 days. At this time, the sample wasreplenished with fresh tissue and the incubation was continued for 4additional days at which time, the assays were scored for insectstunting and mortality.

FIGS. 1 and 2 show the results of insect feeding assays performed usingembryo tissue transformed with the silencing construct DNAs listed inTables 5 and 8. Each symbol corresponds to insect mortality scoresaveraged over the 4 replicate assays for each event. Controls correspondto feeding assays conducted using non-transgenic soybean embryo tissue.For all of the constructs, several transgenic events could be foundwhich gave insect mortality scores greater than the controls. For someconstructs, more than 50% of the events produced insect mortality at arate significantly greater than controls. Variation in apparent efficacyfrom event to event is to be expected due to variation in constructexpression with random integration of the construct DNA in the soybeangenome.

Example 11 Assay of Transgenic Soybean Plants for Efficacy AgainstSouthern Green Stinkbug

Silencing constructs can be stably expressed in insect feeding tissuefor efficacy testing of transgenic plants against southern greenstinkbug. The DNA constructs described in Example 8 can be used for thispurpose. These consist of trait gene hairpin or miRNA gene cassettesboth of which are constitutively regulated by a soybean ubiquitinpromoter-5′UTR-Intron1 fragment. Similar constructs can be built usingother constitutive promoters as provided for example by soybeanelongation factor 1 alpha (ACUP01009998) or arabidopsis ubiquitin(L05399.1) genes. Alternatively, tissue specific expression and in someembodiments seed-preferred promoters can be produced by the use of seedstorage protein promoters including those provided by thebeta-conglycinin-alpha (Genbank accession GU723691), Kunitz trypsininhibitor 3 (AF233296), or the glycinin-1 (AB353075.1) genes. To produceseed specific hairpin constructs (i.e. long dsRNA constructs and miRNAconstructs), entry clones, generated as described in Example 8 above,are combined in an LR clonase reaction with a variant of the destinationvector, pKB499, modified to contain a seed storage protein promoter inplace of the Gm-Ubiquitin promoter. This first LR reaction generates thepromoter-hairpin-terminator cassette. The final plant expressionconstruct is produced by a second LR reaction in which the entirehairpin cassette is moved into a vector (PHP25224) which provides aplant selectable marker gene (herbicide resistant acetolactate synthase)for stable transformation experiments. For assembly of tissue specificmiRNA constructs, the procedure outlined in Example 8 would be followedwith final cloning of the artificial miR159 segment into a suitableplant expression vector that provides regulatory sequences of any one ofthe above seed storage protein promoters.

For biolistic transformation of soybean embryos as described in Example5, a single DNA fragment containing both the trait gene and the plantselectable marker gene is prepared by restriction enzyme digestionfollowed by gel purification of restricted pDNA. In the case of bothconstitutive and tissue specific silencing constructs, 10 μg of plasmidDNA is used in 0.15 mL of the specific enzyme mix described below.Plasmids are digested with AscI (100 units) in NEBuffer 4 (20 mMTris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mMdithiothreitol, pH 7.9), 100 μg/mL BSA, and 5 mM beta-mercaptoethanol at37° C. for 3 hrs. The resulting DNA fragments are separated by gelelectrophoresis on 1% agarose gel and the DNA fragment containing thetrait gene-selectable marker gene cassettes are cut from the agarosegel. DNA is purified from the agarose using Qiagen's Quick Spinextraction method following the manufacturer's suggested protocol. Goldparticles are coated with purified DNA fragments and used for biolisticintroduction of DNA into soybean embryo cultures using the procedureoutlined in Example 5.

First generation transgenic plants can be assayed for insecticidalactivity in individual plant cages. When the plant has started toproduce green pods approximately 1-2 inches in length, plants areremoved to individual bug tent cages (BioQuip, CA). The cage is infestedwith 50 newly emerged second instar southern green stinkbugs (Nezaraviridula). The nymphs are allowed to feed for 1 week at which time acount of surviving insects is performed. Counts are facilitated by usingan aspirating device with removable vials and caps to collect insectsand a hand held counting device to count each insect as it is aspirated.Counts can later be verified by freezing the sample and counting againunder magnification where a measure of growth can also be performed oncollected insects. Fully grown insects equivalent to controls are givena score of 0. Insects demonstrating 20-60% stunting are given a scoreof 1. Insects demonstrating 60-100% stunting (size equivalent tooriginal infested insects) are given a score of 2 and dead insects arescored 3. Selected plants demonstrating high insecticidal activity arerecovered from the tents, treated with Marathon insecticide, andreturned to growth chambers or greenhouses to complete the reproductivephase and seed production.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. An isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) thenucleotide sequence comprising any one of SEQ ID NOS: 279, 302, 281,304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291,292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344,305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298,299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328 or a complementthereof; (b) the nucleotide sequence comprising at least 90% sequenceidentity to any one of SEQ ID NOS279, 302, 281, 304, 280, 283, 282, 303,278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263, 17, 30,34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308, 309,310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321, 322,323, 324, 325, 326, 327 or 328 or a complement thereof, wherein saidpolynucleotide encodes a silencing element having insecticidal activityagainst a Pentatomidae plant pest; (c) the nucleotide sequencecomprising at least 19 consecutive nucleotides of any one of SEQ ID NOS:279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288,289, 290, 291, 292, 17, 30, 34, 14, 18 or 263 or a complement thereof,wherein said polynucleotide encodes a silencing element havinginsecticidal activity against a Pentatomidae plant pest; and, (d) thenucleotide sequence that hybridizes under stringent conditions to thefull length complement of the nucleotide sequence of a), wherein saidstringent conditions comprise hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.1× SSC at 60° C. to 65° C., whereinsaid polynucleotide encodes a silencing element having insecticidalactivity against a Pentatomidae plant pest.
 2. The isolatedpolynucleotide of claim 1, wherein said Pentatomidae plant pest is a N.viridula plant pest.
 3. An expression cassette comprising a heterologouspolynucleotide of claim 1 operably linked to a seed-preferred promoter.4. The expression cassette of claim 3, wherein said polynucleotide isexpressed as a double stranded RNA.
 5. The expression cassette of claim3, wherein said polynucleotide comprise a silencing element which isexpressed as a hairpin RNA.
 6. The expression cassette of claim 5,wherein the silencing element comprises, in the following order, a firstsegment, a second segment, and a third segment, wherein a) said firstsegment comprises at least about 19 nucleotides having at least 90%sequence complementarity to a target sequence set forth in SEQ ID NOS:279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288,289, 290, 291, 292, 17, 30, 34, 14, 18 or 263; b) said second segmentcomprises a loop of sufficient length to allow the silencing element tobe transcribed as a hairpin RNA; and, c) said third segment comprises atleast about 19 nucleotides having at least 85% complementarity to thefirst segment.
 7. The expression cassette of claim 6, wherein saidtarget sequence comprises the sequences set forth any one of SEQ ID NOS:284, 285, 286, 287, 288, 289, 290, 291, 292, 337, 338, 339, 340, 341,342, 343 or 344 or a sequence having at least 90% sequence identity toSEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 337, 338, 339,340, 341, 342, 343 or
 344. 8. The expression cassette of claim 6,wherein said expression cassette comprises any one of SEQ ID NOS: 293,294, 295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326,327 or
 328. 9. The expression cassette of claim 3, wherein saidpolynucleotide is flanked by a first operably linked convergent promoterat one terminus of the polynucleotide and a second operably linkedconvergent promoter at the opposing terminus of the polynucleotide,wherein the first and the second convergent promoters are capable ofdriving expression of the polynucleotide.
 10. A host cell comprising theheterologous expression cassette of claim
 3. 11. A plant cell havingstably incorporated into its genome a heterologous polynucleotidecomprising a silencing element operably linked to a seed-preferredpromoter, wherein said silencing element, when ingested by aPentatomidae plant pest, reduces the level of expression of any one ofthe target sequences set forth in SEQ ID NOS: 279, 302, 281, 304, 280,283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18,263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307,308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301,321, 322, 323, 324, 325, 326, 327 or 328 in said Pentatomidae plant pestand thereby controls the Pentatomidae plant pest.
 12. The plant cell ofclaim 11, wherein said silencing element comprises a) a fragment of atleast 19 consecutive nucleotides of SEQ ID NOS: 279, 302, 281, 304, 280,283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30,34, 14, 18 or 263 or a complement thereof; or, b) the nucleotidesequence comprising at least 90% sequence identity to any one of SEQ IDNOS: 279, 302, 281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287,288, 289, 290, 291, 292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340,341, 342, 343, 344, 305, 306, 307, 308, 309, 310, 311, 312, 293, 294,295, 296, 297, 298, 299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or328 or a complement thereof, wherein said silencing element, wheningested by a Pentatomidae plant pest, reduces the level of a targetsequence in said Pentatomidae plant pest and thereby controls thePentatomidae plant pest.
 13. The plant cell of claim 12, wherein thePentatomidae plant pest is a N. viridula plant pest.
 14. The plant cellof claim 12, wherein said silencing element comprises the sequences setforth in any one of SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291,292, 305, 306, 307, 308, 309, 310, 311, 312, 17, 30, 34, 337, 338, 339,340, 341, 342, 343 or 344 or a complement thereof.
 15. The plant cell ofclaim 11, wherein said plant cell comprises the expression cassette ofclaim
 9. 16. The plant cell of claim 11, wherein said silencing elementexpresses a double stranded RNA.
 17. The plant cell of claim 11, whereinsaid silencing element expresses a hairpin RNA.
 18. The plant cell ofclaim 17, wherein said polynucleotide comprising the silencing elementcomprises, in the following order, a first segment, a second segment,and a third segment, wherein a) said first segment comprises at leastabout 19 nucleotides having at least 90% sequence complementarity to atarget sequence set forth in SEQ ID NOS: 279, 302, 281, 304, 280, 283,282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291, 292, 14, 18, 263,17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308,309, 310, 311, 312, 293, 294, 295, 296, 297, 298, 299, 300, 301, 321,322, 323, 324, 325, 326, 327 or 328; b) said second segment comprises aloop of sufficient length to allow the silencing element to betranscribed as a hairpin RNA; and, c) said third segment comprises atleast about 19 nucleotides having at least 85% complementarity to thefirst segment.
 19. The plant cell of claim 11, wherein said plant cellis from a monocot.
 20. The plant cell of claim 19, wherein said monocotis maize, barley, millet, wheat or rice.
 21. The plant cell of claim 11,wherein said plant cell is from a dicot.
 22. The plant cell of claim 21,wherein said plant is soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton.
 23. A plant or plant part comprising aplant cell of claim
 11. 24. A transgenic seed from the plant of claim23, wherein said transgenic seed comprises said heterologouspolynucleotide comprising said silencing element.
 25. A method ofcontrolling a Pentatomidae plant pest comprising feeding to aPentatomidae plant pest a composition comprising a silencing element,wherein said silencing element, when ingested by said Pentatomidae plantpest, reduces the level of expression of any one of the targetPentatomidae plant pest sequences set forth in SEQ ID NOS: 279, 302,281, 304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290,291, 292, 17, 30, 34, 14, 18 or 263 and thereby controls thePentatomidae plant pest.
 26. The method of claim 25, wherein saidsilencing element comprises a) a fragment of at least 19 consecutivenucleotides of SEQ ID NOS: 279, 302, 281, 304, 280, 283, 282, 303, 278,284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34, 14, 18 or 263or a complement thereof; or, b) the nucleotide sequence comprising atleast 90% sequence identity to any one of SEQ ID NOS: 279, 302, 281,304, 280, 283, 282, 303, 278, 284, 285, 286, 287, 288, 289, 290, 291,292, 14, 18, 263, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343, 344,305, 306, 307, 308, 309, 310, 311, 312, 293, 294, 295, 296, 297, 298,299, 300, 301, 321, 322, 323, 324, 325, 326, 327 or 328 or a complementthereof, wherein said silencing element, when ingested by a Pentatomidaeplant pest, reduces the level of a target sequence in said Pentatomidaeplant pest and thereby controls the Pentatomidae plant pest.
 27. Themethod of claim 26, wherein said Pentatomidae plant pest comprises a N.viridula plant pest.
 28. The method of claim 26, wherein said silencingelement comprises the sequence set forth in any one of SEQ ID NOS: 284,285, 286, 287, 288, 289, 290, 291, 292, 305, 306, 307, 308, 309, 310,311, 312, 17, 30, 34, 337, 338, 339, 340, 341, 342, 343 or 344 or acomplement thereof.
 29. The method of claim 25, wherein said compositioncomprises a plant or plant part having stably incorporated into itsgenome a polynucleotide comprising said silencing element, wherein saidsilencing element is operably linked to a seed-preferred promoter. 30.The method of claim 25, wherein said silencing element comprises a) apolynucleotide comprising the sense or antisense sequence of thesequence set forth in SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290,291, 292, 17, 30, 34, 14, 18, 263, 337, 338, 339, 340, 341, 342, 343,344, 305, 306, 307, 308, 309, 310, 311 or 312 or a complement thereof;or, b) a polynucleotide comprising the sense or antisense sequence of asequence having at least 95% sequence identity to the sequence set forthin SEQ ID NOS: 284, 285, 286, 287, 288, 289, 290, 291, 292, 17, 30, 34,14, 18, 263, 337, 338, 339, 340, 341, 342, 343, 344, 305, 306, 307, 308,309, 310, 311 or 312 ora complement thereof.
 31. The method of claim 25,wherein said silencing element expresses a double stranded RNA.
 32. Themethod of claim 25, wherein said silencing element comprises a hairpinRNA.
 33. The method of claim 32, wherein said polynucleotide comprisingthe silencing element comprises, in the following order, a firstsegment, a second segment, and a third segment, wherein a) said firstsegment comprises at least about 20 nucleotides having at least 90%sequence complementarity to the target polynucleotide; b) said secondsegment comprises a loop of sufficient length to allow the silencingelement to be transcribed as a hairpin RNA; and, c) said third segmentcomprises at least about 20 nucleotides having at least 85%complementarity to the first segment.
 34. The method of claim 29,wherein said silencing element is flanked by a first operably linkedconvergent promoter at one terminus of the silencing element and asecond operably linked convergent promoter at the opposing terminus ofthe polynucleotide, wherein the first and the second convergentpromoters are capable of driving expression of the silencing element.35. The method of claim 29, wherein said plant is a monocot.
 36. Themethod of claim 35, wherein said monocot is maize, barley, millet, wheator rice.
 37. The method of claim 29, wherein said plant is a dicot. 38.The method of claim 37, wherein said plant is soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton.